Synthesis and composition of amino acid linking groups conjugated to compounds used for the targeted imaging of tumors

ABSTRACT

The present disclosure relates to compounds that are useful as near-infrared fluorescence probes, wherein the compounds include i) a pteroyl ligand that binds to a target receptor protein, ii) a dye molecule, and iii) a linker molecule that comprises an amino acid or derivative thereof. The disclosure further describes methods and compositions for making and using the compounds, methods incorporating the compounds, and kits incorporating the compounds.

RELATED APPLICATIONS

The present patent application is related to and claims the prioritybenefit of U.S. Provisional Patent Application Ser. No. 61/791,921,filed Mar. 15, 2013, the content of which is hereby incorporated byreference in its entirety into this disclosure.

FIELD DISCLOSURE

The present disclosure is in the area of diagnostics. This disclosureprovides methods of synthesizing and utilizing amino acid linking groupsthat are conjugated to a compound used for the targeted imaging oftumors. Conjugation of the amino acid linking groups increasespecificity and detection of the compound. Methods and compositions foruse thereof in diagnostic imaging are contemplated.

BACKGROUND OF THE DISCLOSURE

Surgical removal of malignant disease constitutes one of the most commonand effective therapeutic for primary treatment for cancer. Resection ofall detectable malignant lesions results in no detectable return of thedisease in approximately 50% of all cancer patients' and may extend lifeexpectancy or reduce morbidity for patients in whom recurrence of thecancer is seen. Not surprisingly, surgical methods for achieving morequantitative cytoreduction are now receiving greater scrutiny.

Resection of all detectable malignant lesions results in no detectablereturn of the disease in approximately 50% of all cancer patients andmay extend life expectancy or reduce morbidity for patients in whomrecurrence of the cancer is seen. Given the importance of totalresection of the malignant lesions, it is beneficial to ensure that themalignant lesions are accurately and completely identified.Identification of malignant tissue during surgery is currentlyaccomplished by three methods. First, many tumor masses and nodules canbe visually detected based on abnormal color, texture, and/ormorphology. Thus, a tumor mass may exhibit variegated color, appearasymmetric with an irregular border, or protrude from the contours ofthe healthy organ. A malignant mass may also be recognized tactilely dueto differences in plasticity, elasticity or solidity from adjacenthealthy tissues. Finally, a few cancer foci can be locatedintraoperatively using fluorescent dyes that flow passively from theprimary tumor into draining lymph nodes. In this latter methodology,fluorescent (sentinel) lymph nodes can be visually identified, resectedand examined to determine whether cancer cells have metastasized tothese lymph nodes.

Despite the recognition of the importance of removal of tumor and theavailability of certain identification techniques for visualizing tumormass, many malignant nodules still escape detection, leading to diseaserecurrence and often death. Thus, there is a need for improved tumoridentification. This motivation has led to introduction of two newapproaches for intraoperative visualization of malignant disease. In thefirst, a quenched fluorescent dye is injected systemically into thetumor-bearing animal, and release of the quenching moiety by atumor-specific enzyme, pH change, or change in redox potential isexploited to selectively activate fluorescence within the malignantmass. In the second approach, a fluorescent dye is conjugated to atumor-specific targeting ligand that causes the attached dye toaccumulate in cancers that over-express the ligand's receptor. Examplesof tumor targeting ligands used for this latter purpose include folicacid, which exhibits specificity for folate receptor (FR) positivecancers of the ovary, kidney, lung, endometrium, breast, and colon, andDUPA, which can deliver attached fluorescent dyes selectively to cellsexpressing prostate-specific membrane antigen (PSMA), i.e. prostatecancers and the neovasculature of other solid tumors. Beneficially, onefolate-targeted fluorescent dye (folate-fluorescein or EC17) has beenrecently tested intra-operatively in human ovarian cancer patients. Inthis study, ˜5× more malignant lesions were removed with the aid of thetumor-targeted fluorescent dye than without it, and all resectedfluorescent lesions were confirmed by pathology to be malignant.

Conventional fluorescent techniques use probes in the visible lightspectrum (˜400-600 nm), which is not optimal for intra-operativeimage-guided surgery as it is associated with a relatively high level ofnonspecific background light due to collagen in the tissues. Hence thesignal to noise ratio from these conventional compounds is low.Moreover, the absorption of visible light by biological chromophores, inparticular hemoglobin, limits the penetration depth to a fewmillimeters. Thus tumors that are buried deeper than a few millimetersin the tissue may remain undetected. Moreover ionization equilibrium offluorescein (pKa=6.4) leads to pH-dependent absorption and emission overthe range of 5 to 9. Therefore, the fluorescence of fluorescein-baseddyes is quenched at low pH (below pH 5).

For example, the potential use of EC17 dye for a more widespread use inoptical imaging for the characterization and measurement diseased tissuein a clinical setting has been hampered by the major drawback of thatthe attached dye (fluorescein) emits fluorescence in the visible range.This makes EC17 and related dyes poor for in vivo use in tissues becausetissues typically autofluoresce strongly in the visible range, and lightpenetrates tissue poorly. Moreover, EC17(folate-ethelenediamine-fluorescein isothiocynate) consists a thiourealinker. It is well known that thiourea compounds have low shelf life dueto the instability of the thiourea linkage. Thus, a compound such asEC17 is not optimal for use in optical imaging because of thisunstability and the related decomposition of the decomposition ofthiourea bridge.

The combination of light absorption by hemoglobin in the visible lightspectrum (<600 nm) and water and lipids in the IR range (>900 nm),offers an optical imaging window from approximately 650-900 nm in whichthe absorption coefficient of tissue is at a minimum. A suitablealternative to dyes that emit light in the visible range would be todevelop dyes that can be used in the near infra red (NIR) range becauselight in the near infrared region induces very little autofluorescenceand permeates tissue much more efficiently. Another benefit to near-IRfluorescent technology is that the background from the scattered lightfrom the excitation source is greatly reduced since the scatteringintensity is proportional to the inverse fourth power of the wavelength.Low background fluorescence is necessary for highly sensitive detection.Furthermore, the optically transparent window in the near-IR region (650nm to 900 nm) in biological tissue makes NIR fluorescence a valuabletechnology for in vivo imaging and subcellular detection applicationsthat require the transmission of light through biological components.

While the use of light in the NIR range for deeper tissue imaging ispreferable to light in the visible spectrum, the NIR imaging dyescurrently used in the art suffer from a number of challenges anddisadvantages such as a susceptibility to photobleach, poor chemicalstability, absorbance and emission spectra that fall within the samerange as many physiological molecules (resulting in high backgroundsignal and autofluorescence). Moreover, most of the NIR dyes are notstable during the synthesis, especially conjugating to a ligand with anamine linker, leading to multiple unwanted side products. Therefore,taking ligand-targeted NIR imaging agent for clinic can be expensive.Thus, current imaging methods that utilize NIR fluorescent probes arenot effective in deep tissue imaging (>5 mm from the surface), inquantifying fluorescence signal in mammalian tissues, or in productioncost that increase preclinical-to-clinical translational time.

Two promising approaches to fluorescence-guided surgery are currentlyunder intense investigation for use in the clinic. In one method, anactivatable NIR fluorescent probe, which is minimally fluorescent in thesteady state due to its proximity to an attached quencher, becomeshighly fluorescent upon release of the quencher in malignant tissue. Oneof the most commonly used release mechanisms involves incorporation of apeptide sequence between the dye and the quencher that can bespecifically cleaved by a tumor-enriched protease (i.e. cathepsins,caspases and matrix metalloproteinases). A major advantage of thisstrategy lies in the absence of fluorescence in tissues that lack theactivating enzyme, allowing tissues along the excretion pathway (e.g.kidneys, bladder, liver) to remain nonfluorescent unless theyfortuitously express the cleaving enzyme. Such tumor-activated NIR dyescan also generate substantial fluorescence in the tumor mass as long asthe malignant lesion is enriched in the cleaving protease and thereleased dye is retained in the tumor. The major disadvantage of thismethodology arises from the poor tumor specificities of many of therelevant hydrolases (most of which are also expressed in healthy tissuesundergoing natural remodeling or experiencing inflammation). Moreover,the abundance of the desired proteases may vary among tumor masses,leading to slow or no activation of fluorescence in some malignantlesions and rapid development of fluorescence in others.

Thus, there remains a need for a dye substance that can be used tospecifically target diseased tissue and has increased stability andbrightness for use in vivo for tissue imaging.

BRIEF SUMMARY OF THE DISCLOSURE

This disclosure provides a method for synthesizing amino acid linkinggroups that are conjugated to a compound used for the targeted imagingof tumors and lymph nodes. In certain embodiments, this disclosurerelates to a compound or a salt derivative thereof, that comprises afolate or pteroyl ligand, a linking group, and a fluorescent dye. Incertain embodiments, the linking group can be an amino acid, an isomer,a derivative, or a racemic mixture thereof. In other aspects, thefluorescent dye is selected from the group consisting of LS288, IR800,SP054, S0121, KODAK, S2076 and S0456.

In some aspects, this disclosure provides a method of conjugating anamino acid linking group to a fluorescent dye, wherein the amino acidcan be tyrosine, serine, theronine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers, and the derivativesthereof. In certain embodiments, the amino acid, isomers, or thederivatives thereof, contain an —OH, —NH₂, or —SH functional group thatupon addition of the fluorescent dye in slight molar excess produces theconjugation of fluorescent group with the amino acid, isomer, or thederivatives thereof. In other embodiments, the amino acid, isomers, orthe derivatives thereof, contains an —OH functional group that uponsynthesis generates an ether bond with the fluorescent dye thatincreases the brightness and detection of the compound. In someembodiments, this disclosure relates to the conjugation of the aminoacid linking group with the fluorescent dye, wherein the amino acid,isomers, or the derivatives thereof, contains an —SH, —SeH, —PoH, or—TeH functional group that upon synthesis generates a C—S, C—Se, C—Po,or C—Te bond with the fluorescent dye. In some aspects, this disclosurerelates to the conjugation of the amino acid linking group to afluorescent dye that has an absorption and emission maxima between about500 nm and about 900 nm. In other aspects, the amino acid linking groupis conjugated to a fluorescent dye that has an absorption and emissionmaxima between about 600 nm and about 800 nm.

In additional embodiments, this disclosure provides a method forconjugating the amino acid linking group to a folate ligand, wherein theamino acid linking group is tyrosine, serine, theronine, lysine,arginine, asparagine, glutamine, cysteine, selenocysteine, isomers orthe derivatives thereof, and is conjugated to folate through a dipeptidebond. In additional aspects, this disclosure provides a method ofconjugating the linking group with a folate ligand, wherein the linkinggroup is tyrosine, serine, theronine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers, or the derivativesthereof, and is conjugated to folate through a homo-oligopeptide bond.In other embodiments, this disclosure relates to a method of conjugatinga pteroyl ligand to an amino acid linking group, wherein the linkinggroup is tyrosine, serine, theronine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers or the derivatives thereof.In certain aspects, the carboxylic acid of the linking group is bound tothe alpha carbon of any amino acid, hence increasing the specificity ofthe compound for targeted receptors. In some embodiments, the amino acidlinking group contributes specificity to the compound, wherein theobserved binding affinity of the compound to targeted receptors isfolate receptor.

In additional aspects, the compound is highly selective for targeting totumor cells expressing the target receptor.

In other embodiments, this disclosure relates to the use of a compounddesignated, Pte-Tyr-S0456 (OTL-0038) for image guided surgery, tumorimaging, lymph node imaging, inflammatory diseases, atherosclerosis,infection diseases, forensic applications, mineral applications, dental,gel staining, DNA sequencing, nerve staining, or plastic surgery. Inother aspects, the Pte-Tyr-S0456 derivative can be Pte-D-Tyr-S0456,Pte-homoTyr-S0456, Pte-beta-homo-Tyr-S0456, Pte-(NMe)-Tyr-S0456,Pte-Tyr(OMe)-S0456, Pte-Tyr(OBn)-S0456, Pte-NHNH-Tyr-OAc-S0456, salts,or derivatives thereof.

In other aspects, this disclosure provides a method of synthesizing thecompound, wherein a protecting group is used to avoid undesiredreactivity with groups other than the amino groups that might generateunwanted compounds. The methods provided in this disclosure produce afinal compound with a yield of over 98% purity.

In certain aspects, this disclosure relates to a compound used for thetargeted imaging of tumors, wherein the compound could be used forresearch, diagnostic, or therapeutic purposes. In other embodiments,this disclosure provides a composition comprising an imaging compoundand a pharmaceutically acceptable carrier, excipient, diluents, orsalts.

In other aspects, this disclosure relates to a compound which has aformula selected from the group consisting of:

and a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 Folate receptor-targeted 1^(st) generation folate-NIR dyeconjugates.

FIG. 2 Binding isotherms of 1^(st) generation folate-NIR conjugates.Binding curves of folate-NIR dye conjugates to folate receptorexpressing KB cells. The targeted conjugates are DyLight 680(triangles), Alexa Fluor 750 (diamonds), and IR800CW (circles).

FIG. 3 Fluorescent images of mice with metastatic disease (experimentalmodel) 4 hours following intravenous injection of 1^(st) generationfolate-NIR dye conjugates. Fluorescent and white light image overlays ofintact (A-D) and surgically opened (a-d) tumor-bearing mice. Athymicnude mice with FR-expressing metastatic L1210A tumors were injectedintravenously with 10 nmol folate-DyLight 680 (Na) or folate-DyLight 750(B/b) and imaged 4 hours later.

FIG. 4: H&E analysis of tissue resected during sequential tumordebulking surgery. A-D: Fluorescent and white light image overlays ofL1210A metastatic tumor-bearing mice 4 hours following tail veininjection with 10 nmol folate-IR800CW. A: Whole body image. B: Openedchest cavity. C: After removal of primary tumor. D: After removal of allsecondary nodules. a-d: H&E staining of a: Healthy control lung, b:Primary tumor, c: Secondary tumor nodule. d: Residual tissue.

FIG. 5 Folate receptor-targeted 2^(nd) generation folate-NIR dyeconjugates

FIG. 6 Binding isotherms (competition studies with radiolabeled folicacid. This assay will provide binding affinity and specificity forfolate receptor at the same time) of folate-linkers and 2^(nd)generation folate-NIR dye conjugates to cultured cancer cells. FIG. 6ABinding curves of folate-EDA-dye conjugates to folate receptorexpressing KB cells. FIG. 6B Binding curves of folate-Lys-NIR dyeconjugates to folate receptor expressing KB cells.

FIG. 7 Fluorescent images and ex vivo tissue biodistribution of 2^(nd)generation folate-NIR conjugates. FIG. 7A-C Fluorescent images of nudemice with KB tumor xenografts 2 hours following intravenous injection of10 nmol folate-NIR conjugate. FIG. 7A Folate-EDA-NIR conjugatesadministered mouse group imaged individually. FIG. 7B Head-to-headcomparison of folate-EDA-NIR conjugate administered mice. FIG. 7CFolate-Lys-NIR conjugates administered mouse group imaged individually.FIG. 7D ex vivo tissue biodistribution of animals administered withfolate-NIR conjugate, FA-EDA-LS288. FIG. 7E ex vivo tissuebiodistribution of animals administered with folate-NIR conjugate,FA-EDA-IR800. FIG. 7F ex vivo tissue biodistribution of animalsadministered with folate-NIR conjugate, FA-EDA-ZW800. FIG. 7G ex vivotissue biodistribution of animals administered with folate-NIRconjugate, FA-EDA-Kodak. FIG. 7H ex vivo tissue biodistribution ofanimals administered with folate-NIR conjugate, FA-Lys-LS288. FIG. 7I exvivo tissue biodistribution of animals administered with folate-NIRconjugate, FA-Lys-IR800. FIG. 7J ex vivo tissue biodistribution ofanimals administered with folate-NIR conjugate, FA-Lys-ZW800. FIG. 7K exvivo tissue biodistribution of animals administered with folate-NIRconjugate, Pte-Lys-LS288. FIG. 7L ex vivo tissue biodistribution ofanimals administered with folate-NIR conjugate, Pte-DHDADS-LS288.

FIG. 8 Folate receptor-targeted 3^(rd) generation NIR dye conjugates

FIG. 9 Rational of Pte-L-Tyr-S0456 NIR dye conjugate. Chemical structureof Pteroyl-Tyr-S0456 (OTL-0038) with four beneficial functionalities.a=pteroic acid as a targeting molecule; b=α-carboxylic acid fromtyrosine for tumor specificity and improve binding affinity for folatereceptor; c=phenolic moiety from tyrosine to enhance (bright)fluorescence intensity; d=near-IR fluorescent probe. Therefore, tyrosineacts as part of ligand, linker, and near-IR dye. In other word, tyrosineis a linker that improves the binding affinity and specificity of ligand(pteroic acid). It also enhances the brightness of the NIR dye.

FIG. 10A Monitoring of reaction progress of Pte-Tyr-S0456 (OTL-0038).

FIG. 10B Monitoring of reaction progress of folate-EDA-IR800CW by LC/MS.Pte-Tyr-S0456 gave 99% pure desired product with over 98% yield whereasfolate-EDA-IR800CW furnished multiple byproducts with 30-40% of desiredproduct.

FIG. 11A Whole body fluorescent images of nude mice with KB tumorxenografts 2 hours following intravenous injection of 10 nmol folatereceptor targeted-NIR conjugate, Pte-Tyr-S0456 (overlay of Fluorescentand white light images). FIG. 11B ex vivo tissue biodistribution ofconjugates following harvesting tissues previously imaged mice.

FIG. 12A Head-to-head comparison of Pte-L-Try-S0456 (OTL-0038) with2^(nd) generation folate-NIR conjugates whole body fluorescent images.FIG. 12B Head-to-head comparison of Pte-L-Try-S0456 (OTL-0038) with2^(nd) generation folate-NIR conjugates ex vivo tissue biodistribution.FIG. 12C Head-to-head comparison of Pte-L-Try-S0456 (OTL-0038) with2^(nd) generation folate-NIR conjugates tumor and kidney images 2h afteradministering Pte-Tyr-S0456 and 2^(nd) generation folate-NIR conjugates(10 nmol) to nude mice. Dissected (sliced) tumors showed homogeneousuptake of the targeted imaging agents in the tumors.

FIG. 13 Comparisons of tumor accumulation and tumor specificity ofPte-Tyr-S0456 with other pteroyl-NIR dye conjugate after administering10 nmol of each conjugates to mice bearing folate receptor positivetumor xenografts.

FIG. 14 depicts the structure of four compounds conjugated with an aminoacid linking group including Pte-Lys-S0456, Pteroyl-Cys-S0456,Pte-Ser-S0456, and Pte-4-amino-L-Pro-S0456.

FIG. 15 depicts the relative binding affinity of OTL-0038, OTL-0039(D-Isomer of OTL-0038), and folic acid for folate receptors. FIG. 15A isa plot which depicts the binding curve of each compound for folatereceptors. FIG. 15B is a table illustrating the binding affinity andrelative binding affinity of all three compounds.

FIG. 16A depicts the whole body fluorescence image of nude mice with KBtumor xenografts injected with OTL-0039. Mice were intravenouslyinjected with 10 nmol of OTL-0039 in phosphate buffered saline (100 μL).After 2.5 hours, animals were euthanized by CO₂ asphyxiation. Whole bodyimaging experiments were then performed using a Caliper IVIS Lumina IIImaging Station with Living Image 4.0 software.

FIG. 16B illustrates tissue biodistribution of mice injected withOTL-0039 in FIG. 5A, 2.5 hours following injection of compound.Following whole body imaging animals were dissected, and select tissues(heart, lung, liver, spleen, kidneys, stomach, small intestine, largeintestine, muscle, skin, and tumor) were analyzed for fluorescenceactivity using IVIS imager as before.

FIG. 17 depicts a table that summarizes tumor uptake of OTL-0038. Tissuebiodistribution was analyzed in mice injected with increasing amounts ofOTL-0038, ranging from 0.3-90 nmol. Data analysis of biodistribution wasexamined 2.5 hours post injection.

FIG. 18 illustrates the tissue biodistribution of mice injected withincreasing amounts of OTL-0038. The compound concentrations ranging from0.3-90 nmol was administered to mice intravenously. Data analysis ofbiodistribution was examined 2.5 hours post injection. FIG. 18A Tissuebiodistribution of mice injected with 0.3 nmol of OTL-0038. FIG. 18BTissue biodistribution of mice injected with 1 nmol of OTL-0038. FIG.18C Tissue biodistribution of mice injected with 3 nmol of OTL-0038.FIG. 18D Tissue biodistribution of mice injected with 10 nmol ofOTL-0038. FIG. 18E Tissue biodistribution of mice injected with 30 nmolof OTL-0038. FIG. 19F Tissue biodistribution of mice injected with 60nmol of OTL-0038. FIG. 18G Tissue biodistribution of mice injected with90 nmol of OTL-0038.

FIG. 19 illustrates the whole body fluorescence imaging of nude micewith KB tumor xenografts injected with 1 nmol of OTL-0038. Thisdemonstrates that we need very low concentration of OTL-0038 to imagetumor due to its high affinity for FR and higher brightness of the dye.After 2.5 hours, animals were euthanized by CO₂ asphyxiation. Whole bodyimaging experiments were then performed using a Caliper IVIS Lumina IIImaging Station with Living Image 4.0 software.

FIG. 20A depicts the whole body fluorescence image of mice bearing tumorxenografts negative for folate receptors. Whole body imaging wasperformed 2.5 hours after administration of 10 nmol of OTL-0038.

FIG. 20B illustrates invasive tumor and kidney uptake of OTL-0038, byfolate receptor—negative tumor xenografts and folate receptor—positivekidneys. Data analysis was performed 2.5 hours post injection.

FIG. 21 depicts a three step reaction schematic for solution phasesynthesis of imaging compounds.

FIG. 22 depicts a two step reaction schematic for solid phase synthesisof imaging compounds.

FIG. 23 presents whole-body fluorescence images of mice injected with 10nmol of Pte-Tyrosine Analogues-S0456 2 hours post injection. FIG. 23AWhole-body fluorescence images of mice injected with 10 nmol of OTL-00432 hours post injection. FIG. 23B Whole-body fluorescence images of miceinjected with 10 nmol of OTL-0044 2 hours post injection. FIG. 23CWhole-body fluorescence images of mice injected with 10 nmol of OTL-00452 hours post injection. FIG. 23D Whole-body fluorescence images of miceinjected with 10 nmol of OTL-0046 2 hours post injection. FIG. 23EWhole-body fluorescence images of mice injected with 10 nmol of OTL-00472 hours post injection. FIG. 23F Whole-body fluorescence images of miceinjected with 10 nmol of OTL-0048 2 hours post injection.

FIG. 24 presents tissue biodistribution of Pte-Tyrosine Analogues-S04562 hours post injection. FIG. 24A presents tissue biodistribution ofOTL-0043 2 hours post injection. FIG. 24B presents tissuebiodistribution of OTL-0044 2 hours post injection. FIG. 24C presentstissue biodistribution of OTL-0045 2 hours post injection. FIG. 24Dpresents tissue biodistribution of OTL-0046 2 hours post injection. FIG.24E presents tissue biodistribution of OTL-0047 2 hours post injection.FIG. 24F presents tissue biodistribution of OTL-0048 2 hours postinjection.

FIG. 25A Whole-body fluorescence images of mice injected with 10 nmol ofOTL-0038 (Pte-Tyr-50456) 2 hours post injection. Excitation: 745 nm.Emission: 830 nm. FIG. 25B Whole-body fluorescence images of miceinjected with 10 nmol of OTL-0053 (Pteroyl-Lys-S0456) 2 hours postinjection. Excitation: 745 nm. Emission: 830 nm. FIG. 25C Whole-bodyfluorescence images of mice injected with 10 nmol of OTL-0054(Pteroyl-Cys-S0456) 2 hours post injection. Excitation: 745 nm.Emission: 830 nm.

FIG. 26A demonstrates tissue biodistribution of OTL-0038 (Pte-Tyr-S0456)2 hours post injection. Excitation: 745 nm. Emission: 830 nm. FIG. 26Bdemonstrates tissue biodistribution of OTL-0053 (Pteroyl-Lys-S0456) 2hours post injection. Excitation: 745 nm. Emission: 830 nm. FIG. 26Cdemonstrates tissue biodistribution of OTL-0054 (Pteroyl-Cys-S0456) 2hours post injection. Excitation: 745 nm. Emission: 830 nm.

FIG. 27A depicts whole-body fluorescence images of mice injected with 10nmol of OTL-0051 (Pteroyl-Tyr-IRD28) 2 hours post injection. FIG. 27Bdepicts whole-body fluorescence images of mice injected with 10 nmol ofOTL-0052 (Pteroyl-Tyr-Kodak) 2 hours post injection. FIG. 27C depictshalf-body fluorescence images of mice injected with 10 nmol of OTL-0051(Pteroyl-Tyr-IRD28) 2 hours post injection.

FIG. 27D depicts half-body fluorescence images of mice injected with 10nmol of OTL-0052 (Pteroyl-Tyr-Kodak) 2 hours post injection.

FIG. 28A depicts tissue biodistribution of OTL-0051 (Pteroyl-Tyr-IRD28)2 hours post injection. FIG. 28B depicts tissue biodistribution ofOTL-0052 (Pteroyl-Tyr-Kodak) 2 hours post injection.

DETAILED DESCRIPTION OF THE DISCLOSURE

Surgery is one of the best therapies for all the solid tumors, such asprostate, ovarian, lung, breast, colon, and pancreatic cancer. Whilesurgery is effective in 50% of patients with solid tumors in the US,chemo- and radiotherapy alone are effective in less than 5% of allcancer patients. Over 700,000 patients undergo cancer surgery every yearin the US and 40% of surgical patients have a recurrence of locoregionaldisease within 5 years. Despite major advances in the oncology fieldover the last decade, there remain significant hurdles to overcome inthe field. For example, it remains difficult to achieve completeresection of the primary tumor with negative margins, removal of thelymph nodes harboring metastatic cancer cells and identification ofsatellite disease. Achieving improvements in these three cases not onlyimproves disease clearance but also guides decisions regardingpostoperative chemotherapy and radiation. While non-targeted fluorescentdyes have been shown to passively accumulate in some tumors, theresulting tumor-to-background ratios are often poor and the boundariesbetween malignant and healthy tissues can be difficult to define.Although ligand targeted fluorescence dyes (e.g., EC17: Folate-EDA-FITC)have been used for imaging a tissue, those dyes have been ineffective asthey would not penetrate deep tissue and hence only identified thespecific cells on the surface of a tissue rather than deeper within thetissue sample. In addition, it has been shown that the excitation andemission spectra of these previous fluorescence dyes was such that itproduced significant background noise such that the targeted tissue wasnot easily detected. In addition, as discussed in the background above,fluorescein-based dyes have the disadvantages that of low shelf-lifestability. EC17 easily decomposes as a result of the instability of thethiourea bridge in that compound. In addition, as EC17 uses fluoresceinwhich has the drawback of a relatively high level of nonspecificbackground noise from collagen in the tissues surrounding the imagingsite. Moreover, the absorption of visible light by biologicalchromophores, in particular hemoglobin, further limits the usefulness ofdyes that incorporate fluorescein. This means that conventional dyescannot readily detect tumors that may be buried deeper than a fewmillimeters in the tissue. Furthermore, fluorescence from fluorescein isquenched at low pH (below pH 5)

In order for a dye material to be useful in detecting and guidingsurgery or providing other tissue imaging it is important to overcomethese drawbacks.

Several criteria were considered in preparation of conjugates includingnear infrared dyes. Ease of synthesis and chemical stability wereprimary chemical attributes. Spectral properties, such as absorption andemission spectra and quantum yield, were considered. Several biologicalproperties were evaluated, such as binding affinity in cell studies,whole body animal imaging using mice with tumors, and biodistribution.Specifically for biodistribution several aspects were consideredincluding dead mice after 2 hours per oral distribution, live miceimaging and dose escalation. Finally, safety considerations were takenincluding Maximum Tolerance Dose (MTD), ImmunoHistoChemical (IHC)analysis, and general clinical pathology analysis.

The present disclosure provides pteroyl conjugates of near infrared dyesthat are stable, fluoresce in the infrared range, and penetrate deepwithin targeted tissue to produce a specific and bright identificationof areas of tissue that express folate receptor. More specifically, thepteroyl conjugates are linked to the near infrared dyes through an aminoacid linker. Even more specifically, it has been found that where theamino acid linker is tyrosine or a derivative of tyrosine, the intensityof the fluorescence of the dye is maintained or even enhanced.

An amino acid is defined as including an amine functional group linkedto a carboxylic acid functional group, and a side-chain specific to eachamino acid. An alpha amino acid is any compound of the general formulaR⁵CH(NH₂)COOH (α-amino acid), wherein R⁵ is selected from the groupconsisting of H or any known amino acid side chain.

A beta amino acid is defined as including an amine functional grouplinked at a beta carbon and a carboxylic acid functional group linked atthe alpha carbon. A beta homo amino acid is defined as including anamine functional group linked at a beta carbon, a carboxylic acidfunctional group linked at the alpha carbon and a side-chain starting ateither the alpha carbon or the beta carbon wherein the side-chain isbound to another amino acid.

Naturally occurring amino acids can be divided into the following fourgroups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polaramino acids, and (4) neutral nonpolar amino acids. Representative aminoacids within these various groups include, but are not limited to: (1)acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; (2) basic (positively charged) amino acids such asarginine, histidine, and lysine; (3) neutral polar amino acids such asglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine; and (4) neutral nonpolar (hydrophobic) amino acids such asalanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine.

Conserved substitution for an amino acid within a naturally occurringamino acid sequence can be selected from other members of the group towhich the naturally occurring amino acid belongs. For example, thealiphatic side chains group of amino acids is glycine, alanine, valine,leucine, and isoleucine. Conserved substitution of naturally occurringamino acid valine includes use of glycine, alanine, leucine, orisoleucine.

The aliphatic-hydroxyl side chain group of amino acids is serine andthreonine. The amide-containing side chain group of amino acids isasparagine and glutamine. The aromatic side chain group of amino acidsis phenylalanine, tyrosine, and tryptophan. The basic side chain groupof amino acids is lysine, arginine, and histidine. The sulfur-containingside chain group of amino acids having is cysteine and methionine.Examples of naturally conservative amino acids substitutions are: valinefor leucine, serine for threonine, phenylalanine for tyrosine, lysinefor arginine, cysteine for methionine, and asparagine for glutamine.

In preferred embodiments, it is shown herein that such pteroylconjugates specifically target to tumor cells within a tissue. Moreover,the intensity of the fluorescence in greater than the intensity ofpreviously observed with other near infrared dyes that are targeted withfolate for folate receptor positive tumors. This increased intensityallows the targeting and clear identification of smaller areas ofbiological samples (e.g., smaller tumors) from a tissue being monitored.In addition, the increased intensity of the compounds of the presentdisclosure provides the added advantage that lower doses/quantities ofthe dye can be administered and still produces meaningful results. Thus,the compounds of the present disclosure lead to more economical imagingtechniques. Moreover, there is an added advantaged that a lower dose ofthe compounds of the disclosure as compared to conventional imagingcompounds minimizes the toxicity and other side effects that areattendant with administration of foreign materials to a body.

Furthermore, identification of small tumors will lead to a more accurateand more effective resection of the primary tumor to produce negativemargins, as well as accurate identification and removal of the lymphnodes harboring metastatic cancer cells and identification of satellitedisease. Each of these advantages positively correlates with a betterclinical outcome for the patient being treated.

In specific experiments, it was found that use of amino acids other thantyrosine as the linker resulted in loss of near infrared fluorescence.For example, see discussion of Scheme I. Specifically note the syntheticpathway lead to undesired by-product 4 as major product that does nothave NIR properties

However, it is contemplated that in addition to tyrosine and tyrosinederivatives, a pteroyl conjugate of a near infrared dye with cysteine orcysteine derivatives also may be useful. Furthermore, it is contemplatedthat a direct linkage of the pteroyl or folate moiety to the dye orlinkage of the dye to pteroic acid or folic acid through an amine linkeralso produces a loss of intensity of the fluorescence from the conjugatewhereas the presence of the tyrosine or tyrosine derivative as thelinking moiety between the pteroyl (targeting moiety) and the nearinfrared dye (the fluorescing moiety) is beneficial to maintain orenhance the fluorescence of the conjugated compound. Tyrosine-basedcompounds of the disclosure do not require an extra amine linker toconjugate the S0456 and further because conjugation through the phenolmoiety of the tyrosine leads to enhanced fluorescence.

The compounds can be used with fluorescence-mediated moleculartomographic imaging systems, such as those designed to detectnear-infrared fluorescence activation in deep tissues. The compoundsprovide molecular and tissue specificity, yield high fluorescencecontrast, brighter fluorescence signal, and reduce backgroundautofluorescence, allowing for improved early detection and moleculartarget assessment of diseased tissue in vivo (e.g., cancers). Thecompounds can be used for deep tissue three dimensional imaging,targeted surgery, and methods for quantifying the amount of a targetcell type in a biological sample.

Compounds

In an aspect the disclosure relates to compounds comprising the formula:Formula (I):

wherein:

X is an amino acid or a derivative thereof, and

Y is a dye that has a fluorescence excitation and emission spectra inthe near infra red range, and said compound maintains or enhances thefluorescence of Y.

In some embodiments, the amino acid or amino acid derivative induces ashift in the electronic emission spectrum, the electronic absorptionspectrum, or both the electronic emission and absorption spectrum,relative to the electronic spectra of the unmodified dye molecule.Suitably, the shift in the electronic spectrum is a bathochromic shift(i.e., shift to longer wavelength/lower frequency) that helps to improvethe detection of the compound in the near infrared (NIR) spectral windowand/or reduce the amount of background signal, autofluorescence,interferences from the tissue surrounding the area being visualized.More specifically, this shift in electronic spectrum is particularlyobserved with NIR dyes that comprise electronegative atoms that areincorporated into the 6-membered ring. Thus, in certain embodiments theamino acid or amino acid (X) derivative comprises an electron-richmoiety such as, for example, oxygen, sulfur, or nitrogen. Non-limitingexamples of such amino acids can include cysteine, methionine,threonine, serine, tyrosine, phenylalanine, tryptophan, histidine,lysine, arginine, aspartic acid, glutamic acid, asparagine, andglutamine, or derivatives thereof.

In embodiments of this aspect, the disclosure provides compounds ofFormulas (I)a, (I)b, (I)c, and (I)d:

wherein the Tyr, Cys, Ser, and Lys groups indicate a tyrosine, acysteine, a serine, and a lysine amino acid residue, respectively, orderivatives thereof, and L is preferably a pteroyl or folate and Rx eachcomprises an independently selected solubilizing group that isoptionally absent.

Wherein the Tyr, Cys, Ser, and Lys groups indicate a tyrosine, acysteine, a serine, and a lysine amino acid residue, respectively, orderivatives thereof, and L is preferably a pteroyl or folate.Preferably, L is pteroyl.

In specific preferred embodiments the disclosure provides a compound ofFormula I(a), wherein Tyr is selected from the group consisting of:

Suitably, the compounds disclosed herein have a maximum light absorptionwavelengths in the near infrared region of between about 650 nm and 1000nm, for example and preferably, at approximately 800 nm.

In specific preferred embodiments, the compounds disclosed hereininclude a ligand (L) that is effective to target the compound to aparticular cell or tissue type and allow for imaging of that targetedcell or tissue. It is preferable the L is either pteroyl moiety orfolate moiety and more preferable that L is pteroyl moiety. However, itis contemplated that the skilled person may use some other ligand L totarget the compounds to a particular cell surface protein or receptorprotein of interest. In specific and preferred embodiments, the ligandcomprises pteroyl:

Synthesis of Compounds

The compounds disclosed herein can be made using conventional methodsknown in the literature. See for example, the dye compounds weresynthesized as previously reported.

However, in specific preferred embodiments, the present disclosureprovides more efficient synthetic methods for generating the compoundsdescribed herein (i.e., Compounds of Formula I). For example, thecompounds having formulae I(a)-I(d) can be prepared in accordance to thegeneral schemes outlined in each of Schemes I, II, and III below.

Scheme I, illustrates a synthetic scheme previously used to generatecompounds of Formula I where the target ligand comprises folate linkedthrough an amino acid (lysine) to the dye molecule. Briefly, the folateligand modified by attachment to the amino group of the amino acid isreacted with a bridged ether derivative of the dye under conditions toyield products (3) and (4). However, it is notable that compound 3 isthe preferred desirably compound but the synthetic pathway lead topresence of undesired by-product 4 as major product that does not haveNIR properties. Moreover, its spectral properties are pH dependant.Thus, this scheme demonstrates the major drawback of ether bridged dyes.In the conventional production of these dyes, 30-60% of the yield is ofthe desired product and whereas 40-70% of the yield is of the undesiredbyproduct.

Scheme II provides a synthetic route that includes only three reactionsteps and provides the product compound (5) in high yields (above 98%).Briefly, the targeting ligand (1) (illustrated in Scheme II with apteroyl group) and an amino acid or amino acid derivative (2) thatoptionally includes protecting groups to avoid undesired reactivity withgroups other than the amino group of the amino acid are mixed in aHATURO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate)}/DIPEA (Diisopropylethylamine)/DMF(dimethylformamide) solvent system and reacted at a room temperature andfor a sufficient time (5 minutes) to allow coupling of (2) through theamino functionality to ligand (1) to provide (3). Compound (3) can beadvantageously precipitated by adding dilute acid to the reactionmixture. More specifically, Compound 3 was precipitated in 1N HCl(hydrochloric acid) to get final compound over 98% purity, in theseembodiments, the costly HPLC or column chromatography steps are avoided.Compound (3) is reacted to remove the protecting groups on the aminoacid portion of the compound by reacting the compound at roomtemperature in TFA (trifluoroacitic acid):water:TIPS(triisopropylsilane) solvent system for provide compound (4). Thecompound 4 was purified by precipitation with diethyl ether ormethyl-t-butyl ether to yield over 98% purity without HPLC (Highperformance liquid chromatography) or column chromatography. Compound(4) is reacted in a basic aqueous system (e.g., NaOH, sodium hydroxide)in order to remove the protecting group functionalities and issubsequently reacted, in slight molar excess, with the dye (S0456) inwater for a time of 15 minutes and at a temperature of 80-100° C. thatallows for coupling between the dye and (4), to yield final compound(5). Compound 5 was precipitated with acetone to give over 98% purePte-Tyr-S0456. When NaOH is used the sodium salt of Pte-Tyr-S0456 isproduced.

Scheme III provides an alternative solid phase synthetic route toproduce the compounds disclosed herein and provide similar yields asdescribed in Scheme II. Briefly, an amino acid bound to a substrate (1)(illustrated in Scheme III below as protected tyrosine attached to aresin bead) is reacted to remove the Fmoc (Fluorenylmethyloxycarbonyl)protecting group in 20% piperidine in DMF, and is subsequently reactedwith the targeting ligand (again illustrated by pteroyl below) inHATU/DIPEA/DMF for a time and at a temperature sufficient to allowcoupling of the ligand to the amine functional group of the amino acidto provide (2). Compound (2) is reacted to remove the substrate and anyprotecting groups on the amino acid in a series of reactions in aTFA:Water:TIPS solvent system to provide (3). Following a similar finalstep as described in Scheme II, compound (3) is reacted in a basicaqueous system in order to remove the protecting group functionalitiesand is subsequently reacted, in slight molar excess, with the dye(S0456) in water for a time and at a temperature that allows forcoupling between the dye and (3), to yield final compound (4).

The above schemes merely illustrate several non-limiting syntheticapproaches by which the compounds disclosed herein may be prepared. Itwill be appreciated that one of skill in the art will be able toidentify and incorporate modifications to the above schemes that wouldprovide other compounds having the physical properties that are withinthe scope of the disclosure. For example, while the above Schemesillustrates folate and pteroyl groups as the targeting ligands of thecompounds disclosed herein, one of skill will appreciate that othertargeting ligands can be readily incorporated into the synthetic schemeand generate alternative compounds of the Formula I. As another example,a one of skill will appreciate that the absorption/emission wavelengthsof the dye portion of the compounds can be modulated by adjusting thelength of the polymethine chain and selecting the appropriate aryl orheteroaryl groups (e.g., indole vs. benzindole) as well as linking aminoacid groups. In a further example, one of skill in the art willrecognize that the extinction coefficient and fluorescence intensity ofthe dye can be varied by adjusting the rigidity of the polymethine chain(e.g., by introducing a ring system into the polymethine chain such ascyclohexene, cyclobutenone, among others) as is generally known in theart. Accordingly, one of skill in the art will be able to modify thesynthesis by selecting the appropriate reagents to make any of thecompounds disclosed herein and optionally being able to vary particularphysical properties of the compounds.

Methods of Use

As noted herein above, there is a need for near infrared dye compoundsthat specifically target to regions within a tissue. This is so that thecompounds may be used in imaging techniques and to assist in thediagnosis and therapeutic intervention of disease. As discussed indetail above, the compounds provided herein are useful as dyes andimaging agents in the NIR region of the light spectrum. As such, thecompounds have broad applicability to any number of imaging, diagnostic,and targeted therapeutic methods.

In specific embodiments, the present disclosure relates to methods thatincorporate at least one of the compounds disclosed herein (e.g., ofFormula I, I(a), I(b), I(c), and/or I(d)). can be used to specificallyand sensitively identify tumors within a tissue. More specifically, theidentified tumors may then be therapeutically resected through surgicalmethods. In this manner, the compounds of the present disclosure may beuseful in fluorescence guided surgical resection of tumors, lymph nodes,and the like. Alternatively, the compounds of the present disclosure mayreadily be used in whole body imaging in which the compound isadministered to a subject and the localization of the fluorescencefacilitates identification of a tumor site.

In this manner, the compounds of the present disclosure can be used forthe in vivo identification of diseased tissue in a subject in needthereof. The disclosure method includes irradiating an in vivo body partof the subject containing diseased tissue with light having at least oneexcitation wavelength in the near infrared range from about 600 nm toabout 1000 nm. Fluorescence emanating from a compound of the presentdisclosure administered to the subject and which has specifically boundto and/or been taken up by the diseased tissue in the body part, inresponse to the at least one excitation wavelength is directly viewed todetermine the location and/or surface area of the diseased tissue in thesubject.

Light having a wavelength range from 600 nm and 850 nm lies within thenear infrared range of the spectrum, in contrast to visible light, whichlies within the range from about 401 nm to 500 nm. Therefore, theexcitation light used in practice of the disclosure diagnostic methodswill contain at least one wavelength of light to illuminates the tissueat the infrared wavelength to excite the compounds in order that thefluorescence obtained from the area having uptake of the compounds ofthe present disclosure is clearly visible and distinct from theauto-fluorescence of the surrounding tissue. The excitation light may bemonochromatic or polychromatic. In this manner, the compounds of thepresent disclosure are advantageous as they eliminate the need for useof filtering mechanisms that would be used to obtain a desireddiagnostic image if the fluorescent probe is one that fluoresces atwavelengths below 600 nm. In this manner, the compounds of the presentdisclosure avoid obscured diagnostic images that are produced as aresult of excitation light of wavelengths that would be reflected fromhealthy tissue and cause loss of resolution of the fluorescent image.

Operating rooms for surgical procedures can be equipped with an overheadlight that produces wavelengths of light in the optical emittingspectrum useful in practice of disclosure diagnostic methods, such as alamps that produce light in the appropriate wavelength. Such a light canbe utilized in the practice of the disclosure diagnostic methods merelyby turning out the other lights in the operating room (to eliminateextraneous light that would be visibly reflected from tissue in the bodypart under investigation) and shining the excitation light of nearinfrared wavelength into the body cavity or surgically created openingso that the fluorescent image received directly by the eye of theobserver (e.g., the surgeon) is predominantly the fluorescent imageemanating from the fluorophore(s) in the field of vision. Lightemanating from a source in the 600 nm and 850 nm range, preferably 750nm-850 nm range would be used in accomplishing the goal of directvisualization by the observer so that light reflecting from the bodypart, other than that from the fluorescing moiet(ies), is minimized oreliminated.

Accordingly, in disclosure diagnostic methods, the diseased tissue (andbound or taken-up targeting construct) is “exposed” to the excitationlight (e.g, by surgically created opening or endoscopic delivery of thelight to an interior location. The disclosuredisclosed method isparticularly suited to in vivo detection of diseased tissue located atan interior site in the subject, such as within a natural body cavity ora surgically created opening, where the diseased tissue is “in plainview” (i.e., exposed to the human eye) to facilitate a procedure ofbiopsy or surgical excision of the area that has been highlighted byuptake of the compounds of the present disclosure. As the preciselocation and/or surface area of the tumor tissue are readily determinedby the uptake of the compounds of the present disclosure, the methodsemploying the compounds of the present disclosure provide a valuableguide to the surgeon, who needs to “see” in real time the exactoutlines, size, etc. of the mass to be resected as the surgery proceeds.

Thus, in specific embodiments, the present disclosure entails opticalimaging of a biological tissue that expresses a folate receptor bycontacting the tissue with a composition comprising compounds of thepresent disclosure (e.g., compounds of Formula I) and allowing time forthe compound in the composition to distribute within the tissue andinteract with the site of folate receptor. After a sufficient time forsuch interaction has passed, the tissue is illuminated with anexcitation light to cause the compound in the composition to fluoresce.The fluorescence is then detected as and where such fluorescence isobserved is an area that contains the folate receptor.

In like manner, the compounds of the present disclosure are used toidentify a target cell type in a biological sample by contacting thebiological sample with such compounds for a time and under conditionsthat allow for binding of the compound to at least one cell of thetarget cell type. The bound compound is then optically detected suchthat presence of fluorescence of the near infrared wavelength emanatingfrom the bound, targeted compound of the present disclosure indicatedthat the target cell type is present in the biological sample. Thismethod thus provides an image of the targeted cell type in the tissuebeing assessed. Most preferably, the targeted cell type is a tumor cellor a lymph node to which a tumor cell has spread.

These methods advantageously provide an improved method of performingimage guided surgery on a subject as the administration of a compositioncomprising the compound of the disclosure under conditions and for atime sufficient for said compound to accumulate at a given surgical sitewill assist a surgeon in visualizing the tissue to be removed.Preferably the tissue is a tumor tissue and illuminating the compoundthat has been taken up by the tissue facilitates visualization of thetumor by the near infrared fluorescence of the compound using infraredlight. With the aid of the visualization facilitated by the targeting ofthe compound of the disclosure to the tumors site, surgical resection ofthe areas that fluoresce upon excitation by infrared light allows animproved and accurate removal of even small tumors.

It should be understood that in any of the surgical methods of thedisclosure the compounds of the present disclosure may be administeredbefore the surgical incision takes place or even after the surgicalcavity and site of the tumor has been revealed by the surgery.

If the putative diseased site is a natural body cavity or surgicallyproduced interior site, an endoscopic device can be optionally used todeliver the excitation light to the site, to receive fluorescenceemanating from the site within a body cavity, and to aid in formation ofa direct image of the fluorescence from the diseased tissue. Forexample, a lens in the endoscopic device can be used to focus thedetected fluorescence as an aid in formation of the image. As usedherein, such endoscope-delivered fluorescence is said to be “directlyviewed” by the practitioner and the tissue to which the targetingconstruct binds or in which it is taken up must be “in plain view” tothe endoscope since the light used in the disclosure diagnosticprocedure will not contain wavelengths of light that penetrate tissue,such as wavelengths in the near infrared range. Alternatively, theexcitation light may be directed by any convenient means into a bodycavity or surgical opening containing a targeting construct administeredas described herein and the fluorescent image so produced can bedirectly visualized by the eye of the observer without aid from anendoscope. With or without aid from any type of endoscopic device, thefluorescent image produced by the disclosure method is such that it canbe viewed without aid of an image processing device, such as a CCDcamera, TV monitor, photon collecting device, and the like.

It is contemplated that the diagnostic or imaging methods of the presentdisclosure allow the surgeon/practitioner to contemporaneouslysee/view/visualize diseased or abnormal tissue through a surgicalopening to facilitate a procedure of biopsy or surgical excision. As thelocation and/or surface area of the diseased tissue are readilydetermined by the diagnostic procedure of the disclosure employing thecompounds described herein, the disclosure method is a valuable guide tothe surgeon, who needs to know the exact outlines, size, etc. of themass, for example, for resection as the surgery proceeds. In particular,it is noted that the compounds of the disclosure fluorescence in thenear infrared range to a greater intensity than those previouslydescribed. As such, advantageously, it is contemplated that less of thecompound will be needed to achieve diagnostic imaging. In addition, thecompounds of the present disclosure penetrate deep into the tumor andhence the disclosure advantageously allows a greater accuracy that thetumor has been removed.

The present disclosure provides methods for utilizing a diagnosticprocedure during surgery in a subject in need thereof by administeringto the subject a composition comprising a compound of the presentdisclosure and irradiating an in vivo body part of the subjectcontaining diseased tissue with light having at least one excitationwavelength in the range from about 600 nm to about 850 nm, directlyviewing fluorescence emanating from a targeting construct administeredto the subject that has specifically bound to and/or been taken up bythe diseased tissue in the body part, wherein the targeting constructfluoresces in response to the at least one excitation wavelength,determining the location and/or surface area of the diseased tissue inthe subject, and removing at least a portion of the tumor tissue.

In yet another embodiment, the present disclosure provides methods forin vivo diagnosis of tumor tissue in a subject in need thereof. In thisembodiment, the disclosure method comprises contacting samples of tumorcells obtained from the subject in vitro with a plurality of detectablylabeled compounds, each of which binds to or is selectively taken up bya distinct tumor type, determining which of the compounds is bound to ortaken up by the sample tumor cells, administering a diagnosticallyeffective amount of at least one biologically compatible fluorescingtargeting construct containing a compound of the present disclosure thathas been determined to bind to and/or be taken up by the sample tumorcells and a fluorophore responsive to at least one wavelength of lightin the range from about 600 nm to about 850 nm, and diagnosing thelocation and/or surface area of the tumor tissue in the in vivo bodypart by directly viewing fluorescence emanating from the targetingconstruct bound or taken up in the tumor tissue upon irradiation thereofwith light providing the at least one excitation wavelength for thefluorescent targeting construct.

In some embodiments, a single type of fluorescent moiety is relied uponfor generating fluorescence emanating from the irradiated body part(i.e., from the fluorescent targeting construct that binds to or istaken up by diseased tissue) and subjecting the targeting construct witha source of light from the near infrared spectrum.

In other embodiments, it is contemplated that a plurality. (i.e., two,three, four, or more) targeting constructs are used to obtain adiagnostic image. Such additional targeting constructs may be additionalcompounds of the present disclosure distinct from the first suchcompound. Alternatively, the additional targeting constructs maycomprise the dyes described herein but with the pteroyl moiety beingreplaced by a ligand for another receptor other than folate receptor. Instill other embodiments, the additional targeting moieties may be otherfluorescing targeting constructs (e.g., antibodies, or biologicallyactive fragments thereof, having attached fluorophores) that bind toother receptors or antigens on the tumor or tissue (e.g., a site ofatherosclerosis, infection, cardiovascular diseases, neurodegenerativediseases, immunologic diseases, autoimmune diseases, respiratorydiseases, metabolic diseases, inherited diseases, infectious diseases,bone diseases, and environmental diseases or the like) to be imaged. Anyadditional targeting moiety that specifically targets the tumor orspecific site on the tissue may be used provided that it is specific forthe site to be monitored. The purpose of the additional fluorescingtargeting construct is to increase the intensity of fluorescence at thesite to be monitored thereby aiding in detection of diseased or abnormaltissue in the body part. For example, a given tumor may have numerousmarkers and in addition to the compounds of the present disclosure acocktail of fluorescent moieties is provided which are specific for thatgiven tumor such that the signal emanating from the tumor is generatedby more than one compound or fluorescent moiety that has targeted andbecome localized to the tumor site of interest.

In practice, the skilled person would administer a compound of thepresent disclosure either alone or as part of a cocktail of targetingdetectable moieties and allow these compounds and targeting moieties tobind to and/or be taken up by any targeting tissue that may be presentat the site under investigation and then provide a supply of the lightsource. Typically, the compounds of the present disclosure and anyadditional targeting moieties will be administered prior to surgery fora time and in compositions that allow the fluorescent compounds of thepresent disclosure as well as any additional fluorescent constructs tobe taken up by the target tissue.

Those of skill in the art will be able to devise combinations ofsuccessively administered fluorescing targeting constructs, each ofwhich specifically binds to the target site. It is preferable that allof the fluorescing targeting constructs used in such cocktails toidentify the target tissue comprise fluorophores that fluoresce withinthe same wavelength band or at the same wave length as does the compoundof the present disclosure (e.g. a fluorescing sensitive to near infraredwavelength of light in the compounds of the present disclosure) tominimize the number of different light sources that need to be employedto excite simultaneous fluorescence from all of the different targetingconstructs used in practice of the disclosure method. However, it iscontemplated that the additional targeting moieties other than thecompounds of the present disclosure may fluorescence in response to theirradiating light at a different color (i.e., has a differentwavelength) than that from the florescent compounds of the presentdisclosure. The difference in the colors of the fluorescence emanatingfrom the compounds of the present disclosure and those of the additionaltargeting compounds may aid the observer in determining the location andsize of the diseased tissue. In some examples, it may be desirable toinclude fluorophores in targeting constructs targeted to target normaltissue and the compounds of the present disclosure to target diseasedtissue such that the contrast between the diseased tissue and normaltissue is further enhanced to further aid the observer in determiningthe location and size of the target tissue. The use of such additionalfluorophores and targeting agents in addition to the compounds of thepresent disclosure provides the advantage that any natural fluorescenceemanating from normal tissue is obscured by the fluorescence emanatingfrom fluorophore(s) in supplemental targeting constructs targeted to thenormal tissue in the body part. The greater the difference in colorbetween the fluorescence emanating from normal and target tissue, theeasier it is for the observer to visualize the outlines and size of thetarget tissue. For instance, targeting a fluorescing targeting constructcomprising a fluorophore producing infrared light from the compounds ofthe present disclosure to the target tissue (i.e., abnormal tissue) anda fluorophore producing green light to healthy tissue aids the observerin distinguishing the target tissue from the normal tissue. Those ofskill in the art can readily select a combination of fluorophores thatpresent a distinct visual color contrast.

The spectrum of light used in the practice of the disclosure method isselected to contain at least one wavelength that corresponds to thepredominate excitation wavelength of the targeting construct, or of abiologically compatible fluorescing moiety contained within thetargeting construct. Generally the excitation light used in practice ofthe disclosure method comprises at least one excitation wavelength oflight in the near infrared wavelength range from about 600 nm to about850 nm

However, when a combination of targeting ligands that fluoresce atdifferent wavelengths is used in practice of the disclosure, thespectrum of the excitation light must be broad enough to provide atleast one excitation wavelength for each of the fluorophores used. Forexample, it is particularly beneficial when fluorophores of differentcolors are selected to distinguish normal from diseased tissue, that theexcitation spectrum of the light(s) include excitation wavelengths forthe fluorophores targeted to normal and target tissue.

As noted herein the compounds of the present disclosure are specificallytargeted to the folate receptor by way of pteroyl or folate ligand beingpart of the compounds of the present disclosure. In embodiments where anadditional targeting moiety is used, the targeting construct of such anadditional targeting moiety is selected to bind to and/or be taken upspecifically by the target tissue of interest, for example to an antigenor other surface feature contained on or within a cell thatcharacterizes a disease or abnormal state in the target tissue. As inother diagnostic assays, it is desirable for the targeting construct tobind to or be taken up by the target tissue selectively or to an antigenassociated with the disease or abnormal state; however, targetingconstructs containing ligand moieties that also bind to or are taken upby healthy tissue or cell structures can be used in the practice of thedisclosure method so long as the concentration of the antigen in thetarget tissue or the affinity of the targeting construct for the targettissue is sufficiently greater than for healthy tissue in the field ofvision so that a fluorescent image representing the target tissue can beclearly visualized as distinct from any fluorescence coming from healthytissue or structures in the field of vision.

For example, colon cancer is often characterized by the presence ofcarcinoembryonic antigen (CEA), yet this antigen is also associated withcertain tissues in healthy individuals. However, the concentration ofCEA in cancerous colon tissue is often greater than is found in healthytissue, so an anti-CEA antibody could be used as a ligand moiety in thepractice of the disclosure. In another example, deoxyglucose is taken upand utilized by healthy tissue to varying degrees, yet its metabolism inhealthy tissues, except for certain known organs, such as the heart, issubstantially lower than in tumor. The known pattern of deoxyglucoseconsumption in the body can therefore be used to aid in determination ofthose areas wherein unexpectedly high uptake of deoxyglucose signals thepresence of tumor cells.

The disease or abnormal state detected by the disclosure method can beany type characterized by the presence of a known target tissue forwhich a specific binding ligand is known. For example, various heartconditions are characterized by production of necrotic or ischemictissue or production of artherosclerotic tissue for which specificbinding ligands are known. As another illustrative example, breastcancer is characterized by the production of cancerous tissue identifiedby monoclonal antibodies to CA15-3, CA19-9, CEA, or HER2/neu. It iscontemplated that the target tissue may be characterized by cells thatproduce either a surface antigen for which a binding ligand is known, oran intracellular marker (i.e. antigen), since many targeting constructspenetrate the cell membrane. Representative disease states that can beidentified using the disclosure method include such various conditionsas different types of tumors, bacterial, fungal and viral infections,and the like. As used herein “abnormal” tissue includes precancerousconditions, necrotic or ischemic tissue, and tissue associated withconnective tissue diseases, and auto-immune disorders, and the like.Further, examples of the types of target tissue suitable for diagnosisor examination using the disclosure method include cardiac, breast,ovarian, uterine, lung, endothelial, vascular, gastrointestinal,colorectal, prostatic tissue, endocrine tissue, and the like, as well ascombinations of any two or more thereof.

Simply by way of example, antigens for some common malignancies and thebody locations in which they are commonly found are known to those ofskill in the art, and targeting ligands, such as antibodies or for theseantigens or indeed ligands where the antigens are receptors are known inthe art. For example, CEA (carcinoembryoinc antigen) is commonly foundin tumors from the colon, breast and lung; PSA (prostate specificantigen, or sometimes referred to as prostate specific membrane antigen(PSMA)) is specific for prostate cancer; CA-125 is commonly found intumors of ovarian cancer origin, CA 15-3, CA19-9, MUC-1, Estrogenreceptor, progesterone receptor and HER2/neu are commonly found inbreast cancer tumors, alpha-feto protein is found in both testicularcancer and hepatic cancer tumors, beta-human chorionic gonadotropin isfound testicular cancer and choriocarcinoma, both estrogen receptor andprogesterone receptor also are found in uterine cancer tumors andepidermal growth factor receptor is commonly found in tumors frombladder cancer. Other tumor specific ligands and markers are well knownto those of skill in the art. In preferred embodiments, the presentdisclosure employs folate or pteroyl moieties for targeting the folatereceptor and PMSA target moieties for targeting the dyes to prostatecancer cells.

It is contemplated that any of these commonly known markers of tumorscan be targeted either using the dyes described herein (by switching outthe pteroyl moiety for a moiety that specifically targets these markers)or alternatively, these markers can be targeted in addition and incombination with the folate receptor that is being targeted using thecompounds of the present disclosure. As discussed previously, it may beparticularly advantageous to have targeting moieties to severaldifferent markers on a given tumor to serve as a diagnostic cocktail inwhich several markers are targeted to more brightly and clearlyvisualize the tumor.

In addition to chemical compounds, the targeting moieties in suchcocktails may include a protein or polypeptide, such as an antibody, orbiologically active fragment thereof, preferably a monoclonal antibody.The supplemental fluorescing targeting construct(s) used in practice ofthe disclosure method may also be or comprise polyclonal or monoclonalantibodies tagged with a fluorophore. The term “antibody” as used inthis disclosure includes intact molecules as well as functionalfragments thereof, such as Fab, F(ab′)2, and Fv that are capable ofbinding the epitopic determinant. Methods of making these fragments areknown in the art. (See for example, Harlow & Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1988,incorporated herein by reference). As used in this disclosure, the term“epitope” means any antigenic determinant on an antigen to which theparatope of an antibody binds. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

In addition to antibodies, the cocktails may comprise compounds in whichthe ligand moiety attached to the fluorescent targeting construct isselected from among the many biologically compatible compounds that bindwith specificity to receptors and/or are preferentially taken up bytumor cells, and can be used as the ligand moiety in the disclosuretargeting constructs. Compounds that are preferentially “taken up” bytumor cells may enter the cells through surface or nuclear receptors(e.g., hormone receptors), pores, hydrophilic “windows” in the celllipid bilayer, and the like.

Illustrative of this class of compounds to target tumors aresomatostatin, somatostatin receptor-binding peptides, deoxyglucose,methionine, and the like. Particularly useful somatostatinreceptor-binding peptides are a long-acting, octapeptide analog ofsomatostatin, known as octreotide(D-phenylalanyl-L-cysteinyl-L-phenylala-nyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]--L-cysteinamide cyclic(2→7)-disulfide), lanreotide, an oral formulation of octreotide, P829,P587, and the like. Somatostatin-binding peptides are disclosed in U.S.Pat. No. 5,871,711, and methods for linking such peptides covalently toa radioisotope through their carboxyl terminal amino acid under reducingconditions are disclosed in U.S. Pat. No. 5,843,401, which are bothincorporated herein by reference in their entireties. One of skill inthe art can readily adapt such teachings for the preparation offluorescence-sensitive somatostatin receptor-binding peptides bysubstituting the fluorescing moieties of this disclosure in the place ofa radioisotope.

Somatostatin and somatostatin receptor-binding peptides are particularlyeffective for use as the tumor-targeting ligand moiety in the targetingconstruct when the disease state is a neuroendocrine or endocrine tumor.Examples of neuroendocrine tumors that can be diagnosed using thedisclosure method include adenomas (GH-producing and TSH-producing),islet cell tumors, carcinoids, undifferentiated neuroendocrinecarcinomas, small cell and non small cell lung cancer, neuroendocrineand/or intermediate cell carcinomas, neuroendocrine tumors of ovary,cervix, endometrium, breast, kidney, larynx, paranasal sinuses, andsalivary glands, meningiomas, well differentiated glia-derived tumors,pheochromocytomas, neuroblastomas, ganglioneuro(blasto)mas,paragangliomas, papillary, follicular and medullary carcinomas inthyroid cells, Merkel cell carcinomas, and melanomas, as well asgranulomas and lymphomas. These tumor cells are known to havesomatostatin receptors and can be targeted using somatostatin orsomatostatin receptor binding peptides as the tumor-targeting ligand inthe disclosure fluorescent targeting construct. Vasointestinal peptide(VIP), which is used in VIP receptor scintigraphy (I. Virgolini, Eur J.Clin. Invest. 27(10):793-800, 1997, is also useful in the disclosuremethod for diagnosis of small primary adenocarcinomas, liver metastasesand certain endocrine tumors of the gastrointestinal tract.

Another molecule illustrative of the tumor-targeting ligands that arepreferentially taken up by tumors is deoxyglucose, which is known to bepreferentially taken up in a variety of different types of tumors.Illustrative of the types of tumors that can be detected usingdeoxyglucose as the tumor-targeting ligand include melanoma, colorectaland pancreatic tumors, lymphoma (both HD and NHL), head and neck tumors,myeloma, cancers of ovary, cancer, breast, and brain (high grade andpituitary adenomas), sarcomas (grade dependent), hepatoma, testicularcancer, thyroid (grade dependent) small cell lung cancer, bladder anduterine cancer, and the like.

Yet other tumor-targeting compounds that can be used in cocktails of thepresent disclosure include 1-amino-cyclobutane-1-carboxylic acid andL-methionine. L-methionine is an essential amino acid that is necessaryfor protein synthesis. It is known that malignant cells have alteredmethionine metabolism and require an external source of methionine.

Additional examples of biologically compatible tumor-targeting compoundsthat bind with specificity to tumor receptors and/or are preferentiallytaken up by tumor cells include mammalian hormones, particularly sexhormones, neurotransmitters, and compounds expressed by tumor cells tocommunicate with each other that are preferentially taken up by tumorcells, such as novel secreted protein constructs arising fromchromosomal aberrations, such as transfers or inversions within theclone.

Hormones, including sex hormones, cell growth hormones, cytokines,endocrine hormones, erythropoietin, and the like also serve well astumor targeting moieties. As is known in the art, a number of tumortypes express receptors for hormones, for example, estrogen,progesterone, androgens, such as testosterone, and the like. Suchhormones are preferentially taken up by tumor cells, for example, viaspecific receptors.

The targeting constructs and supplemental targeting constructs used inpractice of the disclosure method can be administered by any route knownto those of skill in the art, such as topically, intraarticularly,intracisternally, intraocularly, intraventricularly, intrathecally,intravenously, intramuscularly, intraperitoneally, intradermally,intratracheally, intracavitarily, and the like, as well as by anycombination of any two or more thereof.

The most suitable route for administration will vary depending upon thedisease state to be treated, or the location of the suspected conditionor tumor to be diagnosed. For example, for treatment of inflammatoryconditions and various tumors, local administration, includingadministration by injection directly into the body part to be irradiatedby the excitation light (e.g., intracavitarily) provides the advantagethat the targeting construct (e.g., fluorescently tagged antibodies) canbe administered in a high concentration without risk of thecomplications that may accompany systemic administration thereof.

The compounds of the present disclosure as well as any additionaltargeting constructs used in diagnostic cocktails comprising thecompounds of the present disclosure are administered in a “effectiveamount” for diagnosis. An effective amount is the quantity of atargeting construct necessary to aid in direct visualization of anytarget tissue located in the body part under investigation in a subject.A “subject” as the term is used herein is contemplated to include anymammal, such as a domesticated pet, farm animal, or zoo animal, butpreferably is a human. Amounts effective for diagnostic use will, ofcourse, depend on the size and location of the body part to beinvestigated, the affinity of the targeting construct for the targettissue, the type of target tissue, as well as the route ofadministration. Local administration of the targeting construct willtypically require a smaller dosage than any mode of systemicadministration, although the local concentration of the targetingconstruct may, in some cases, be higher following local administrationthan can be achieved with safety upon systemic administration.

Since individual subjects may present a wide variation in severity ofsymptoms and each targeting construct has its unique diagnosticcharacteristics, including, affinity of the targeting construct for thetarget, rate of clearance of the targeting construct by bodilyprocesses, the properties of the fluorophore contained therein, and thelike, the skilled practitioner will weigh the factors and vary thedosages accordingly.

The compounds of the present disclosure as well as cocktails comprisingthese compounds can be formulated as a sterile injectable suspensionaccording to known methods using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1-4, butanediol. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed, including synthetic mono- or diglycerides, fatty acids(including oleic acid), naturally occurring vegetable oils like sesameoil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fattyvehicles like ethyl oleate, or the like. Buffers, preservatives,antioxidants, and the like, can be incorporated as required, or,alternatively, can comprise the formulation.

It will be apparent to those skilled in the art that various changes maybe made in the disclosure without departing from the spirit and scopethereof, and therefore, the disclosure encompasses embodiments inaddition to those specifically disclosed in the specification, but onlyas indicated in the appended claims.

The examples that follow are merely provided for the purpose ofillustrating particular embodiments of the disclosure and are notintended to be limiting to the scope of the appended claims. Asdiscussed herein, particular features of the disclosed compounds andmethods can be modified in various ways that are not necessary to theoperability or advantages they provide. For example, the compounds canincorporate a variety of amino acids and amino acid derivatives as wellas targeting ligands depending on the particular use for which thecompound will be employed. One of skill in the art will appreciate thatsuch modifications are encompassed within the scope of the appendedclaims.

EXAMPLES Example 1 Development of Tumor-Targeted Near Infrared Dyes forFluorescence Guided Surgery

Complete surgical resection of malignant disease is the only reliablemethod for intervention in cancer. Unfortunately, quantitative tumorresection is often limited by a surgeon's ability to locate allmalignant disease and distinguish it from healthy tissue. Fluorescenceguided surgery has emerged as a tool to aid surgeons in theidentification and removal of malignant lesions. While non-targetedfluorescent dyes have been shown to passively accumulate in some tumors,the resulting tumor-to-background ratios are often poor and theboundaries between malignant and healthy tissues can be difficult todefine. To circumvent these problems, the present disclosure showsdevelopment of high affinity tumor targeting ligands that bind toreceptors that are over-expressed on cancer cells and deliver attachedmolecules selectively into these cells.

In the present example, use of two tumor-specific targeting ligands(i.e. folic acid that targets the folate receptor (FR) to deliver nearinfrared (NIR) fluorescent dyes specifically to FR-expressing cancers,thereby rendering only the malignant cells highly fluorescent. Thepresent Example shows that all FR-targeted NIR dyes examined bindcultured cancer cells in the low nanomolar range. Moreover, uponintravenous injection into tumor-bearing mice with metastatic disease,these same ligand-NIR dye conjugates render receptor-expressing tumortissues fluorescent, enabling their facile resection with minimalcontamination from healthy tissues.

1A: Materials and Methods

The results shown in the present example were obtained using specificmaterials and methods described herein. It is contemplated that theskilled person may be able to modify these methods, reaction conditions,and test conditions and still produce results that demonstrate theefficacy of the FR-targeted NIR dyes of the present disclosure.

a. Synthesis and Characterization of Folate-NIR Conjugates.

All ligands and linkers were synthesized as previously reported inliterature. After purification, folate-targeting ligands were conjugatedto selected NIR dyes as shown in FIGS. 1, 5, and 8. Dye conjugates werepurified using reverse phase preparative HPLC [Waters, xTerra C18 10 μm;19×250 mm; λ=280 nm; solvent gradient: 0 to 30% or 80% B in 30 min run,A=10 mM NH₄OAc buffer in water (pH=7.0), B=Acetonitrile (ACN)]. Purifiedcompounds were analyzed using LC-MS (ESI) mass spectrometry (Waters,X-Bridge C18 5 μm; 3.0×15 mm).

b. Culture of Folate Receptor Expressing Cells Lines.

L1210A cells were obtained from Dr. Manohar Ratnam and KB cells wereobtained from American Type Culture Collection (ATCC; Rockville, Md.).Cells were cultured in folate deficient 1640 RPMI medium supplementedwith 10% heat inactivated fetal bovine serum (HIFBS), 1% L-glutamine,and 1% penicillin streptomycin (Invitrogen, Carlsbad, Calif.). All celllines were cultured in 5% carbon dioxide, 95% air-humidified atmosphereat 37° C.

c. Analysis of Binding Affinity and Specificity of Folate-NIR DyeConjugates by Fluorometery.

KB (200,000 cells/well in 500 μL) were seeded into 24-well plates andallowed to form monolayers over 48 h. Spent medium in each well wasreplaced with fresh medium (0.5 mL) containing increasing concentrationsof folate-NIR dye conjugate in the presence or absence of 100-foldexcess competing ligand; i.e. folic acid. After incubating for 1 hour at37° C., cells were rinsed with fresh medium (3×0.5 mL), dissolved in 1%aqueous SDS (0.600 mL), and assayed for fluorescence by transfer to aquartz cuvette and analysis of fluorescence emission intensity at eachdye's excitation and emission maximum using a Agilent Technologies CaryEclipse fluorescence spectrophotometer. The conjugate's dissociationconstant (K_(d)) was calculated by plotting fluorescence emission unitsversus the concentration of targeted near infrared dye added usingGraphPad Prism 4.03.

d. In Vivo Mouse Models of Metastasis.

All animal procedures were carried out with the approval of the PurdueAnimal Care and Use Committee. For studies involving FR-expressingtumors, 5-6 week old female DBA/2 mice were purchased from HarlanLaboratories (Indianapolis, Ind.) and placed on folate deficient dietfor two weeks prior to and during each study. Tumor metastases wereinduced by injecting 1×10⁶ L1210A (FR expressing) cells into the leftventricle of the heart using a 30 gauge needle. Tumors were allowed todevelop for 4 weeks, after which the animals were injected intravenouslywith 10 nmols of the desired FR-targeted NIR dye dissolved in 100 μlsaline. After 4 hours, animals were sacrificed by CO₂ asphyxiation andimaged as described below.

e. Fluorescent Imaging of Mice with Metastatic Disease.

Animal imaging experiments were performed using a Caliper Ivis Lumina IIImaging Station with Living Image 4.0 software. Settings for imagingAlexa Fluor 647 and DyLight 680 conjugates: lamp level: high;excitation: 605; emission: Cy5.5; epi illumination; binning: (M) 4;FOV=7.5; f-stop=4; acquisition time=1s. Settings for imaging DyLight 750and IR800CW conjugates: lamp level: high; excitation: 745; emission:ICG; epi illumination; binning: (M) 4, FOV=12.5; f-stop=4; acquisitiontime=1 s.

f. H&E Staining of Normal and Diseased Tissues.

After imaging, organs were dissected and stored in 5 ml formalin andsubmitted to the Purdue Histology & Phenotying Laboratory for H&Estaining. In brief, tissue samples were processed using a SakuraTissue-Tek VIP 6, sectioned using a Thermo Finesse ME microtome andstained with H&E reagent using a Shandon Vari-Stain 24-2 autostainer.H&E stained slides were then imaged using an Olympus BH-2 researchmicroscope with an Olympus DP70 camera.

B. Results

a. Synthesis of Tumor-Targeted NIR Dyes.

For selective tumor targeting, the inventors conjugated commerciallyavailable NIR dyes to folate. Most folate-NIR dye conjugates weresynthesized at high yield and subsequently purified using HPLC tohomogeneity.

b. Binding Affinity and Specificity of Targeted NIR Dyes.

Because the cargo attached to a ligand can often interfere with ligandbinding, it was beneficial to test the binding affinities of thefolate-NIR dye conjugates to FR expressing cancer cells. Bindingaffinities of all conjugates were found to be in the low nanomolar range(FIG. 2), with some variation depending on the attached dye, suggestingthat the linked cargo only mildly influences ligand binding. Thespecificity of folate-NIR dye conjugates for their receptors was alsodetermined in vitro by adding excess folic acid. As seen in FIG. 2,binding was nearly quantitatively inhibited by co-incubation with100-fold molar excess of folic acid.

c. Imaging of Tumor-Targeted NIR Dyes In Vivo.

Prior to evaluation of the tumor specificities of the tumor-targeteddyes in vivo, it was beneficial to compare the intensities of theselected dyes upon excitation through tissue. For this purpose, 1 mL ofphosphate buffered saline containing 100 nM each of dye (Alexa Fluor647, DyLight 680, DyLight 750, IR800CW) was placed in an Eppendorf tube,which in turn was positioned under a 1 cm thick section of fresh porcinemuscle, and the resulting tissues were imaged under the same conditionsin both a Kodak Image Station and IVIS Lumina Imager, only the optimalexcitation and emission wavelengths were always selected for each dye ineach instrument. IR800CW produced the brightest fluorescent signal, withDyLight 750 yielding a signal of intermediate intensity, and Alexa Fluor647 and DyLight 680 displaying the weakest fluorescence.

In order to compare the abilities of the above folate-NIR dye conjugatesto detect metastatic tumor nodules in vivo, a murine model of tumormetastasis was developed that involved intracardiac injection of 10⁶L1210A cells (FR expressing cells) followed by normal husbandry of themice for 4 weeks to allow nascent tumors to grow. Tumor-bearing micewere then treated with 10 nmol of selected folate-NIR dye conjugate viatail vein injection, and mice were euthanized 4 hours later forfluorescence imaging. As seen in FIG. 3, tumor loci could be readilydistinguished, yielding strong contrast between fluorescent cancernodules and adjacent healthy tissues. In some cases, fluorescent tumorscould even be seen in images of intact mice (FIG. 3, top panels),however, due to differences in tumor size, location, and depth, it wasnot possible to unequivocally establish which NIR dye yielded the bestimages in intact animals.

Finally, in order to mimic a live surgical setting, resection offluorescent tumor tissue was performed in stages, with the largestmasses being removed first, and smaller malignant loci being excisedafter more prominent fluorescent masses had been cleared (FIG. 4).Beneficially, removal of the primary masses often revealed secondarymetastases that were not visible prior to the initial rounds of surgeryand would have likely been missed without the aid of the tumor-specificfluorescence. Following these multiple rounds of resection, when allvisible fluorescence had been removed, excised tissues were submittedfor histological analysis, and these studies revealed that allfluorescent nodules were indeed malignant. Beneficially, random samplingof the remaining tissues demonstrated that nonfluorescent regions werenonmalignant (FIG. 4 d), suggesting an apparent quantitative removal ofcancerous lesions with the aid of the tumor-targeted fluorescent dyes.

C. Discussion

A second approach to fluorescence-guided surgery involves conjugation ofan NIR dye to a tumor-specific targeting ligand that binds avidly tocancer cells and clears quantitatively from most healthy tissues.Advantages of this approach include: i) the rapid rate of tumorvisualization, owing to the fact that tumor uptake and normal tissueclearance of the dye can occur within minutes of intravenous injection,ii) the stability of tumor contrast, arising from the fact that theligand-dye conjugates are commonly internalized by the cancer cells viareceptor-mediated endocytosis, iii) the specificity of the fluorescencewhenever the targeted receptor is either absent, weakly expressed, orinaccessible in normal tissues, and iv) the absence of “bleeding” offluorescence from malignant into nonmalignant tissues, due to highaffinity retention of the ligand-dye conjugate on its receptor, creatinghighly defined boundaries that clearly demark the cancer. A disadvantageof the strategy derives from the fact that the ligand-targeted dye isalways fluorescent, even during excretion, preventing imaging of kidneyand bladder tumors until excretion of the dye is complete.

One surprising result from these studies was the smaller size ofmalignant lesions that could be readily detected in vivo. Thus, moredetailed analyses of several sites with punctate metastatic diseaserevealed that cancer cell clusters as small as 50 μm could be visualizedwith use of higher resolution optics. Because clusters of even a fewcells can eventually lead to recurrence of the cancer, the ability todetect and remove even the smallest metastatic lesions could eventuallylead to reduced patient mortality, assuming an appropriate camera can bedesigned.

While most applications of fluorescence-guided surgery likely remain tobe discovered, some uses of the technology can already be envisioned.First, more malignant lesions will potentially be identified andresected due to better visualization of tumor masses. Second, in caseswhere maximal preservation of normal tissues is essential (i.e. cancersof the brain, breast, pancreas, head and neck, etc.), careful shaving offluorescent lesions until no fluorescence remains might enable moreefficient conservation of healthy tissue. Third, pre-operative stagingof cancer patients might eventually be possible via laparoscopicinterrogation of proximal lymph nodes for fluorescent lesions, obviatingthe need for surgery when significant metastases are clearly observedand eliminating the requirement for subsequent surgical sampling ofsentinel lymph nodes when only a single tumor mass is detected.

In conclusion, the present Example demonstrates that tumor-targeted NIRdyes have the potential to reshape standard surgical procedures byimproving visualization of malignant tissues, leading more complete andprecise diseased tissue removal and improved patient outcome.

Example 2 Design and Synthesis of the Optimal Folate ConjugatedNear-Infrared Probe with High Targeting Affinity and Sensitivity forFluorescence Guided Cancer Surgery

Even with the sophisticated tools for tumor identification, manymalignant nodules still escape detection, leading to disease recurrenceand often death. Motivated by a need for improved tumor identification,two new approaches for intraoperative visualization of malignant diseasehave been introduced. In the first, a quenched fluorescent dye isinjected systemically into the tumor-bearing animal, and release of thequenching moiety by a tumor-specific enzyme, pH change, or change inredox potential is exploited to selectively activate fluorescence withinthe malignant mass. In the second, a fluorescent dye is conjugated to atumor-specific targeting ligand that causes the attached dye toaccumulate in cancers that over-express the ligand's receptor. Examplesof tumor targeting ligands used for this latter purpose include folicacid, which exhibits specificity for folate receptor (FR) positivecancers of the ovary, kidney, lung, endometrium, breast, and colon.Beneficially, a folate-targeted fluorescent dye (folate-fluorescein orEC17) has been recently tested intra-operatively in human ovarian cancerpatients. In that study, ˜5× more malignant lesions were removed withthe aid of the tumor-targeted fluorescent dye than without it, and allresected fluorescent lesions were confirmed by pathology to bemalignant.

Unfortunately, a major deficiency with the above clinical study derivedfrom the fact that the attached dye (fluorescein) emits fluorescence inthe visible range, i.e. where autofluorescence is strong and lightpenetrates tissue poorly. Because light in the near infrared (NIR)region induces little autofluorescence and permeates tissue much moreefficiently, the inventors postulated that a more complete tumorresection would be possible if an NIR dye were used to guide thesurgery. Although there are a limited number of commercially availableNIR fluorophores and largely based on the cyanine chemical structurewith specific modifications by each manufacturer (FIG. 6). Fortunately,each of the fluorophore series come as reactive fluorophores that can bereadily conjugated to the protein or ligand of interest for specific invivo targeting through conjugation chemistry. Most of the commerciallyavailable experimental NIR fluorophores are available asN-hydroxysuccinimide (NHS) esters which can be used for fluorophoreconjugation at N-terminal amine. To evaluate and identify the bestfolate conjugated NIR probe for image guided cancer surgery, theinventors have conjugated the photostable NHS ester NIR dyes (compoundsa-c below) with N-terminal amine functionalized folates (Fol-EDA andFol-Lys) yielded the expected NIR probes (1a-c and 2a-c) along with themajor elimination side products (1e-g and 2e-g). In order to get theenhanced yield the more stable NIR dye LS288 NHS ester (d) was utilizedand isolated the folate conjugated NIR dyes 1d and 2d with good yields.The optical properties and photostability of the synthesized folate-NIRprobes 1a-d and 2a-d were characterized.

The fluorescence excitation and emission spectra of all folate-NIRprobes 1a-d and 2a-d (500 □L; 5 □M in PBS) at same concentration showedalmost similar intensity. The cytotoxicity and folate receptor affinityof the probe were investigated by in vitro cell experiments and thetumor-targeting capability was in vivo investigated in five groups ofnude mice bearing FR⁺ KB tumor xenograft also biodistribution wereexamined by using the Lumina II near-infrared fluorescence imagingsystem. Unfortunately, the biodistribution shown synthetically favorablehigh yielding folate-NIR probes of FIG. 6 compound 1d and FIG. 7compound 2d had two fold less brightness of the fluorescence intensityon tumor compare with other folate-NIR probes of FIG. 6 compounds 1a-cand FIG. 7 compounds 2a-c.

  IR800CW,   ZW800,   LMNIR2,   LS288

1a 1b 1c 1d

2a 2b 2c 2d                

 

In view of the structure the beneficial variation was the substitution(S_(NR1)) at the central vinylogous cyclohexe carbon (C(sp²)) by phenolmoiety in NIR dyes a-c and phenyl moiety in NIR dye d also with theircorresponding final folate-NIR probes. These above reasons havemotivated the inventors to develop a new modified folate-NIR probe withphotostable, selective, sensitive and high fluorescent intensity atphysiological pH.

For solving the problem, the overall strategy was to choose S0456 as acommercially available precursor NIR fluorophore should contains fourSO₃H groups for solubility and rigid cyclohexenyl ring in the middle offluorophore for photostability and vinylogous cyclohexe chloride(C(sp²)Cl) to connect phenolic oxygen which will be beneficial for highsensitivity and bright fluorescence for in vivo tumor imaging. Moreover,the structural requirements for folate receptor ligands with folatebinding pocket have been investigated to improve the binding affinityand selectivity of the ligands to the receptor. The crystal structure offolate receptor has not been established and there is an argument thatpteroic acid, a fragment of folic acid lacking the distal glutamylresidue is good enough for binding to the high affinity folate receptor.To determine whether the □-carboxylic acid in glutamyl residue isbeneficial for binding with folate receptor the inventors synthesizedvarious amino acid and non-amino acid pteroyl conjugates and its NIRprobes. Next, the inventors compared all these new modified folate-NIRprobes for in vitro binding affinity with folate receptor positivecancel cells and in vivo imaging with folate receptor positive KB tumor.Interestingly, the inventors did not observe much variation in bindingaffinity but there was a remarkable change in tumor specificity andfluorescence intensity. The study was consistent with all folate-NIRconjugates and suggested □-carboxylic acid in glutamyl residue isbeneficial for specific tumor targeting, uptake and the substitution ofphenolic oxygen on the central vinylogous cyclohexe carbon (C(sp²)) inNIR dye will be beneficial for high fluorescence intensity of the tumortissue. Therefore, it is urgent to develop a simpler and morestraightforward strategy to obtain optimal new modified folate-NIRfluorescent probe for image guided cancer surgery. The inventorsdesigned and synthesized a sensitive, photostable and tumor selectivenew modified folate-NIR fluorescent probe (Pteroyl-Tyr-S0456; FIG. 6)with high fluorescence intensity for a clinical application.

In the present Example, the inventors designed a folatereceptor-targeted near-infrared fluorescence probe (Pteroyl_Tyr_S0456)with strengthened fluorescence intensity and photostability. The hightargeting capability for folate receptor-overexpressed tumors withbright fluorescence intensity was demonstrated. This newPteroyl_Tyr_S0456 conjugation improved the dynamics of the probe in micesubjects and enhanced the targeting capability and sensitivity to FRoverexpressed tumors. Results in this Example demonstrate that this newNIR probe possesses great potential in the diagnosis of early stagetumors.

Example 3 Comparative Analysis of OTL-0001 (FA-EDA-L288), OTL-0002(FA-EDA-IR800), OTL-0003 (FA-EDA-ZW800), and OTL-0004 (FA-EDA-Kodak2)

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude mice (5 weeks old, 18-20 g) were purchased fromHarlan (IN) and maintained on gamma-irradiated folate-deficient specialdiet (Teklad, Wis.) for at least 2 weeks before the start of the study.Animals were housed 5/cage in a barrier, pathogen-free cloaked rack.Autoclaved tap water and food were given as needed. The animals werehoused in a sterile environment on a standard 12 h light-dark cycle forthe duration of the study. Mice were identified individually by earpunch. All animal procedures were approved by Purdue Animal Care and UseCommittee. Animal care and studies were performed according to nationaland international guidelines for the humane treatment of animals.

Whole Body Imaging:

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (2 mice/group) wereintravenously injected with 10 nmol of test article (FA-EDA-LS288,FA-EDA-IR800, FA-EDA-ZW800 and FA-EDA-Kodak2) in phosphate bufferedsaline (100 μL). After 2 h, animals were euthanized by CO₂ asphyxiation.Whole body imaging (intact tumor) experiments were then performed usinga Caliper IVIS Lumina II Imaging Station with Living Image 4.0 software(PerkinElmer Inc, MA). Settings for imaging:—lamp level: medium;excitation: 745 nm; emission: 830 nm; epi illumination; binning: 4 (M),FOV=12.5; f-stop=2; acquisition time=1s.

Tissue Biodistribution:

Following whole body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission: 830 nm; epi illumination; binning:4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Results:

Whole body imaging: As seen in the FIG. 7 a, FA-EDA-LS288, FA-EDA-IR800,and FA-EDA-ZW800 accumulated predominantly in the folate receptorpositive tumors, with no substantial fluorescence activity in the othertissues. However, FA-EDA-Kodak2 did not accumulated in the tumors.Moreover, direct comparison demonstrated that tumor fluorescenceintensity FA-EDA-IR800 injected mice were brighter (higher fluorescenceintensity) than the mice treated with the other folate-conjugated nearIR dyes (FIG. 7 b).

Conclusion:

The brightness and specificity of the conjugates listed from best toworst are as follows: FA-EDA-IR800, FA-EDA-ZW800, FA-EDA-LS288,FA-EDA-Kodak2. The conjugates containing IR800 and ZW800 showed thehighest tumor-accumulated fluorescence while the conjugate containingKodak showed very low specificity for the tumor. The low fluorescenceseen in the Kodak conjugate may be due to the fact that the dye excitesat 800 nm. The IVIS image system does not have a filter to excite at 800nm, so the low fluorescence in the tumors may be due to using a poorexcitation wavelength.

Example 4 Whole Body Imaging and Biodistribution of FolateReceptor-Targeted Near Infrared Dyes

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude mice (5 weeks old, 18-20 g) were purchased fromHarlan (Indianapolis, Ind.) and maintained on gamma-irradiatedfolate-deficient special diet (Teklad, Wis.) for at least 2 weeks beforethe start of the study. Animals were housed 5/cage in a barrier,pathogen-free cloaked rack. Autoclaved tap water and food were given asneeded. The animals were housed in a sterile environment on a standard12 h light-dark cycle for the duration of the study. Mice wereidentified individually by ear punch. All animal procedures wereapproved by Purdue Animal Care and Use Committee. Animal care andstudies were performed according to national and internationalguidelines for the humane treatment of animals.

Whole Body Imaging:

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (2-3 mice/group) wereintravenously injected with 10 nmol of test article in phosphatebuffered saline (100 μL). After 2 hours, animals were euthanized by CO₂asphyxiation. Whole body imaging (intact tumor) experiments were thenperformed using a Caliper IVIS Lumina II Imaging Station with LivingImage 4.0 software (PerkinElmer Inc, MA). Settings for imaging:—lamplevel: medium; excitation: 745 nm; emission: ICG (830 nm); epiillumination; binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Tissue Biodistribution:

Following whole body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, and tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission:ICG (830 nm); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Results:

Whole Body Imaging:

As seen in the FIG. 1, FA-EDA-LS288, FA-EDA-IR800, and FA-EDA-ZW800accumulated predominantly in the folate receptor positive tumors, withno substantial fluorescence activity in the other tissues. Moreover,direct comparison demonstrated that tumor fluorescence intensityFA-EDA-IR800 injected mice were brighter (higher) than the mice treatedwith the other folate-conjugated near IR dyes (FIG. 2).

Tissue Biodistribution:

Analysis of tissue biodistribution was performed on animals under thesame conditions by euthanizing each mouse, removing their organs andimaging them using an IVIS imager. As seen in the FIG. 2, the highestfluorescence intensity was observed in FR-positive tumors and thekidneys. The kidney uptake was anticipated since the apical membrane ofthe proximal tubule of the kidney has been known to express high levelsof folate receptor. Moreover, it's possible that the probes are excretedthrough the kidneys due to their low molecular weights and half-life(most of the folate conjugates have <30 min half-life).

Conclusion:

The brightness and specificity of the conjugates listed from best toworst are as follows: FA-EDA-IR800, FA-EDA-ZW800, FA-EDA-LS288,FA-EDA-Kodak2. The conjugates containing IR800 and ZW800 showed thehighest tumor-accumulated fluorescence while the conjugate containingKodak showed very low specificity for the tumor and low fluorescencecompared to the others.

A. Materials and Methods

The results shown in the present example were obtained using specificmaterials and methods described herein. It is contemplated that theskilled person may be able to modify these methods, reaction conditions,and test conditions and still produce results that demonstrate theefficacy of the FR-targeted NIR dyes of the present disclosure.

a. Synthesis and Characterization of Folate-NIR Conjugates.

Synthesis and characterization of folate NIR conjugates was performedsubstantially as described in Example 1.

b. Relative Binding Affinity of Folate-NIR Conjugates.

(4) to FR.KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates and allowed to form monolayers over a periodof 24 h. Spent medium in each well was replaced with 10 nM [³H]-folatein the presence of increasing concentration (0.1 nM-1 μM) of the testarticle or folic acid in fresh medium (0.5 mL). After incubating for 1hour at 37° C., cells were rinsed with PBS (2×0.5 mL) and 1 Mtrichloroacetic acid (1×0.5 mL) to remove any unbound radioactivematerials. After adding 1% sodium dodecylsulfate in PBS (0.5 mL), cellswere transferred into individual scintillation vials containing Ecolumescintillation cocktail (3.0 mL) and counted in a .liquid scintillationanalyzer. The relative binding affinities were calculated using a plotof cell bound radioactivity versus the concentration of the test articleusing GraphPad Prism 4.

c. In Vivo Mouse Models of Subcutaneous Tumor Xenografts.

Five-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in RPMI medium) on their shoulders. Growth of thetumors was measured in two perpendicular directions every 2 days using acaliper (body weights were monitored on the same schedule), and thevolumes of the tumors were calculated as 0.5×L×W2 (L) longest axis andW) axis perpendicular to L in millimeters). Once tumors reached between400 and 500 mm³ in volume, animals were treated with folatereceptor-targeted NIR dye conjugate (10 nmol) in phosphate bufferedsaline (100 μL). After 2 h, animals were sacrificed by CO₂ asphyxiationand imaged as described below.

d. Fluorescent Imaging of Mice with FR⁺ KB Tumor.

Animal imaging experiments were performed using a Caliper Ivis Lumina IIImaging Station with Living Image 4.0 software. Settings for imagingAlexa Fluor 647 and DyLight 680 conjugates: lamp level: high;excitation: 605; emission: Cy5.5; epi illumination; binning: (M) 4;FOV=7.5; f-stop=4; acquisition time=1 s. Settings for imaging DyLight750 and IR800CW conjugates: lamp level: high; excitation: 745; emission:ICG; epi illumination; binning: (M) 4, FOV=12.5; f-stop=4; acquisitiontime=1 s.

B. Results

a. Synthesis of Tumor-Targeted NIR Dyes.

For selective tumor targeting, the inventors conjugated commerciallyavailable NIR dyes to either folate or pteroate via amide bond formationusing NHS activated dye or Williamson ether synthesis reaction usingchloro-derivative of NIR dye. Pte-aminoacid-NIR, especially tyrosine,were synthesized in very high yield (>98%) without any HPLC or specialpurification technique (by precipitation) with very high purity (>98%).The amino acids refereeing here are tyrosine and its analogues, cystine,serine, lysine, etc. The NIR dyes that we used are S0456, Kodak, S0121,and S2076 (not limited to). Synthesis of ether bridged NIR conjugates offolates such as folate-IR800CW (3) and folate-ZW800 (5), however,resulted in production of prominent side products (FIG. 10B) that neededespecial purification techniques such as HPLC thereby leading to higherproduction cost and increasing time length for preclinical to clinictranslation. This will not only effect for advancement of surgicaloncology but also patients who are waiting for new therapeutic agents.Moreover, higher production cost may indirectly effect for patients andtheir insurance providers due to increasing cost of the drug.Importantly, Pte-Tyr-S0456 reaction did not yield any undesired byproduct and reaction was completed within 15 min with high yield andhigh purity (FIG. 10A).

b. Binding Affinity and Specificity of Folate Receptor-Targeted NIRDyes.

The affinity and specificity of folate- and pteroate-NIR conjugates werefirst evaluated using cancer (KB) cells that overexpressed folatereceptor. The competition studies with tritiated folic acid(radiolabeled folic acid) demonstrated that folate- or pteroate-NIRconjugates not only binding to folate receptor with high affinity (lownanomolar values) but also with high specificity (FIG. 6). Thecompetition studies with tritiated folic acid (radiolabeled folic acid)demonstrated that Pte-Tyr-S0456 binds folate receptor with high affinityand specificity suggesting that conjugation of bulky S0456 moiety viaphenolic oxygen did not compromise the binding of Pte-Tyr to flotereceptor.

c. Imaging of Tumor-Targeted NIR Dyes In Vivo.

In order to compare the abilities of the above folate-NIR dye conjugatesto detect tumors, a xenograft model was developed that involvedimplantation of KB cells subcutaneously (FR expressing cells) followedby normal husbandry of the mice for 4 weeks to allow nascent tumors togrow. Tumor-bearing mice were then treated with 10 nmol of selectedfolate-NIR dye conjugate via tail vein injection, and mice wereeuthanized 2 hours later for fluorescence imaging. As seen in FIG. 7Atumor loci could be readily distinguished, yielding strong contrastbetween fluorescent cancer nodules and adjacent healthy tissues. Mostimportantly, intact fluorescent tumors were even seen in images ofintact mice without opening or harvesting the tumor (FIG. 7A).Head-to-head whole body fluorescence imaging study for 2^(nd) generationfolate receptor targeted NIR agents indicated that folate-IR800CW (3)was competitive (in terms of fluorescent brightness) to all the otherdyes (FIG. 7A, 2^(nd) raw). However, unfortunately, folate-IR800CW (3)was not stable during the synthesis leading to form over 60% ofundesired byproducts. As mentioned before, this will cause for findingespecial purification techniques indicating path for higher productioncost, higher waiting period for clinical translation, and surgeons andpatients will not have access to the drug.

To establish the in vivo specificity, Pte-Tyr-S0456 was administered tomice bearing folate receptor positive tumor xenografts on theirshoulders. In vivo whole body imaging studies demonstrated thatPte-Tyr-S0456 is mainly accumulated in the folate receptor positivetumors and no fluorescence was observed in other tissues (FIG. 11A). Exvivo tissue biodistribution studies indicated that Pte-Tyr-S0456accumulated predominantly in the folate receptor positive tumors with nosubstantial fluorescence activity in other organs except the kidneys(FIGS. 11B, and 12 B). Significant uptake in kidneys was anticipated,since the apical membrane of the proximal tubule of the kidney has beenknown to express high levels of FR. Head-to-head comparison study ofPte-Tyr-50456 with folate-IR800CW, folate-LS288, and folate-ZW800demonstrated that Pte-Tyr-S0456 is competitive, in terms of fluorescentbrightness, to all folate-NIR conjugates (FIGS. 12 A & B). Moreover,fluorescence imaged of sliced (dissected) tumors suggested that bothPte-Tyr-S0456 and all folate-NIR conjugates were accumulatedhomogeneously in all tumor cells even that are buried inside the tumor(FIG. 12C).

Example 5 In Vitro Pharmacology Studies of Folate- and Pteroyl-NIR Dyes

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates (BD Biosciences, CA) and allowed to formmonolayers over a period of 12 h. Spent medium in each well was combinedwith 10 nM of [³H]-folic acid (tritiated folic acid) in the presence ofincreasing concentration (0.1 nM-1 NM) of the test article or folic acid(Sigma-Aldrich, MO) in fresh medium (0.5 mL). After incubating for 1 hat 37° C., cells were rinsed with PBS (3×0.5 mL, Gibco, NY) to removeany unbound radioactive materials. After adding 0.25 M sodium hydroxide(0.5 mL) and incubating for 12 h at 4° C., cells were transferred intoindividual scintillation vials containing Ecolite scintillation cocktail(3.0 mL, MP Biomedicals, OH) and counted in a liquid scintillationanalyzer (Packard). The relative binding affinities were calculatedusing a plot of % cell bound radioactivity versus the log concentrationof the test article using GraphPad Prism 4.

Results:

The dissociation constants (K_(D)) derived from the studies wascalculated to be 30.7 nM, 19.3 Nm, 23.3 nM, 30.6 nM, 50.1 nM, 22.8 nM,30.5 nM, 39.7 nM, 49.6 nM, 30.5 nM, and 8 nM for compoundsOTL-001-OTL-0010 and folic acid respectively. Relative bindingaffinities were calculated to be 0.270, 0.430, 0.356, 0.271, 0.166,0.364, 0.272, 0.209, 0.167, 0.272 and 1 for OTL-0001-OTL-0010 and folicacid respectively. All the test articles competed quantitatively with[³H]-folic acid. Relative binding affinity is defined as the molar ratioof the compound required to displace 50% of [³H]-folic acid bound tofolate receptor on cells; relative affinity of folic acid=1; relativeaffinity<1 indicates weaker affinity for folate receptor; relativeaffinity>1 indicates stronger binding to folate receptor.

Conclusion:

All the compounds have a affinity for folate receptor and they comparemoderately well with the binding affinity of folic acid. All thecompounds competed well with [³H]-folic acid indicating that folatereceptor constitutes the sole binding site on cancer cells and they arehighly specific for folate receptor.

Example 6 In Vitro Pharmacology Studies of OTL-0038 and OTL-0039(D-Isomer of OTL-0038)

Two ligand-NIR conjugates were developed and designated OTL-0038 andOTL-0039. OTL-0038 compound refers to PTE-L-Tyr-S0456, where pteroyl,the ligand is conjugated to L-tyrosine, which is linked to S0456.OTL-0039 is the D-isomer of OTL-0038. The binding affinity and bindingspecificity of both compounds for folate receptors were examined incomparison to folic acid, the conjugate ligand for folate receptors.

A. Material and Methods

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates (BD Biosciences, CA) and allowed to formmonolayers over a period of 12 hours. Spent medium in each well wascombined with 10 nM of [³H]-folic acid (tritiated folic acid) in thepresence of increasing concentration (0.1 nM-1 μM) of the OTL-0039(D-isomer) and OTL-0038 (L-isomer), or folic acid (Sigma-Aldrich, MO) infresh medium (0.5 mL). After incubating for 1 hour at 37° C., cells wererinsed with PBS (3×0.5 mL, Gibco, NY) to remove any unbound radioactivematerials. After adding 0.25 M sodium hydroxide (0.5 mL) and incubatingfor 12 hours at 4° C., cells were transferred into individualscintillation vials containing Ecolite scintillation cocktail (3.0 mL,MP Biomedicals, OH) and counted in a liquid scintillation analyzer(Packard). The relative binding affinities were calculated using a plotof % cell bound radioactivity versus the log concentration of the testarticle using GraphPad Prism 4.

B. Results

The dissociation constants (K_(D)) derived from the studies wascalculated to be 81.8 nM, 10.4 nM, and 7.4 nM for OTL-0039, OTL-0038, orfolic acid respectively. Relative binding affinities were calculated tobe 0.09, 0.71, and 1 for OTL-0039, OTL-0038, and folic acidrespectively. All three test articles competed quantitatively with[³H]-folic acid.

Relative binding affinity is defined as the molar ratio of the compoundrequired to displace 50% of [³H]-folic acid bound to folate receptor oncells; relative affinity of folic acid=1; relative affinity<1 indicatesweaker affinity for folate receptor; relative affinity>1 indicatesstronger binding to folate receptor.

C. Conclusion

OTL-0038 has affinity for folate receptor and it compares well with thebinding affinity of folic acid (10.4 nM Vs 7.4 nM). On the other hand,OTL-0039 has lower affinity for folate receptor when compared to folicacid and OTL-0038. OTL-0038 competed well with [³H]-folic acidindicating that folate receptor constitutes the sole OTL-0038 bindingsite on cancer cells and it is highly specific for folate receptor.

Example 7 Whole Body Imaging and Biodistribution of OTL-0038 andOTL-0039 (D-Isomer of OTL-0038) in Mice Bearing Folate Receptor—PositiveTumor Xenografts

The folate receptor positive tumor uptake of OTL-0038 (PTE-L-Tyr-S0456)and OTL-0039 (PTE-D-Tyr-S0456) was examined to determine how well bothcompounds were taken up by target receptors on tumors. The tissuebiodistribution of the compounds were also examined. Both propertieswere examined in mice two and a half hours following intravenousadministration of the compounds.

A. Material and Methods Cell Culturing and Animal Preparation

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude (nu/nu) mice (5 weeks old, 18-20 g) were purchasedfrom Harlan Laboratories (Indianapolis, Ind.) and maintained ongamma-irradiated folate-deficient special diet (Teklad, Wis.) for atleast 2 weeks before the start of the study. Animals were housed 5/cagein a barrier, pathogen-free cloaked rack. Autoclaved tap water and foodwere given as needed. The animals were housed in a sterile environmenton a standard 12 hour light-dark cycle for the duration of the study.Mice were identified individually by ear punch. All animal procedureswere approved by Purdue Animal Care and Use Committee. Animal care andstudies were performed according to national and internationalguidelines for the humane treatment of animals.

Whole Body Imaging

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (5 mice/group) wereintravenously injected with 10 nmol of OTL-0038 or OTL-0039 in phosphatebuffered saline (100 μL). After 2.5 hours, animals were euthanized byCO₂ asphyxiation. Whole body imaging (intact tumor) experiments werethen performed using a Caliper IVIS Lumina II Imaging Station withLiving Image 4.0 software (PerkinElmer Inc, MA). Settings forimaging:—lamp level: medium; excitation: 745 nm; emission:ICG(indocyanine green); epi illumination; binning: 4 (M), FOV=12.5;f-stop=2; acquisition time=1 s.

Tissue Biodistribution

Following whole body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission: ICG; epi illumination; binning: 4(M), FOV=12.5; f-stop=2; acquisition time=1 s.

B. Results Whole Body Imaging

As seen in the FIG. 14, OTL-0038 accumulated predominantly in the folatereceptor positive tumors, with no substantial fluorescence activity inthe other tissues.

Tissue Biodistribution

Analysis of tissue biodistribution was performed on the same animalsthat were subjected to whole body imaging by euthanizing each mouse,removing their organs and imaging using IVIS imager. As seen in the FIG.15, the highest fluorescence intensity was observed in FR-positivetumors with no accumulation in the other tissues except the kidneys.Uptake of OTL-0038 in the kidneys was anticipated, since the apicalmembrane of the proximal tubule of the kidney has been known to expresshigh levels of folate receptor. Moreover, it is possible that the probesare excreted through the kidneys due to their low molecular weights andhalf-life (most of the folate conjugates have <30 min half-life).

C. Conclusion

OTL-0038 mainly accumulated in folate receptor positive tumor xenograftsand kidneys. All the other normal tissues displayed minimal levels or nouptake, resulting in excellent tumor-to-normal tissue fluorescenceratios.

Example 8 Comparative Analysis of OTL-0038 (L-Isomer) with FolateDerived Near IR Agents

The whole body imaging and tissue biodistribution of OTL-0038 wascompared to folate-LS288, folate-IR800, and folate-ZW800. Thesecompounds were conjugated to folate and commercially availablenear-infrared dyes, LS288, IR800, and ZW800.

A. Material and Methods Cell Culture and Mouse Preparation

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude mice (5 weeks old, 18-20 g) were purchased fromHarlan Laboratories (Indianapolis, Ind.) and maintained ongamma-irradiated folate-deficient special diet (Teklad, Wis.) for atleast 2 weeks before the start of the study. Animals were housed 5/cagein a barrier, pathogen-free cloaked rack. Autoclaved tap water and foodwere given as needed. The animals were housed in a sterile environmenton a standard 12 hour light-dark cycle for the duration of the study.Mice were identified individually by ear punch. All animal procedureswere approved by Purdue Animal Care and Use Committee. Animal care andstudies were performed according to national and internationalguidelines for the humane treatment of animals.

Whole Body Imaging

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (2 mice/group) wereintravenously injected with 10 nmol of test article (OTL-0038,folate-LS288, folate-IR800, folate-ZW800) in phosphate buffered saline(100 μL). After 2.5 h, animals were euthanized by CO₂ asphyxiation.Whole body imaging (intact tumor) experiments were then performed usinga Caliper IVIS Lumina II Imaging Station with Living Image 4.0 software(PerkinElmer Inc, MA). Settings for imaging:—lamp level: medium;excitation: 745 nm; emission: ICG (indocyanine green); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Tissue Biodistribution

Following whole body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission: ICG; epi illumination; binning: 4(M), FOV=12.5; f-stop=2; acquisition time=1 s.

B. Results Whole Body Imaging

As seen in the FIG. 14, OTL-0038 (L-isomer), folate-LS288, folate-IR800,folate-ZW800 accumulated predominantly in the folate receptor positivetumors, with no substantial fluorescence activity in the other tissues.Moreover, direct comparison demonstrated that tumor fluorescenceintensity OTL-0038 injected mice were brighter (higher) than the micetreated with the other folate-conjugated near IR dyes (FIG. 15).

Tissue Biodistribution

Analysis of tissue biodistribution was performed on the same animalsthat were subjected to whole body imaging by euthanizing each mouse,removing their organs and imaging them using an IVIS imager. As seen inthe FIG. 16, the highest fluorescence intensity was observed inFR-positive tumors and the kidneys. The kidney uptake was anticipatedsince the apical membrane of the proximal tubule of the kidney has beenknown to express high levels of folate receptor. Moreover, it's possiblethat the probes are excreted through the kidneys due to their lowmolecular weights and half-life (most of the folate conjugates have <30min half-life).

C. Conclusion

OTL-0038 has beneficial aspects relative to folate-LS288, folate-IR800,and folate-ZW800 in tumor accumulated fluorescence intensity. OTL-0038may be brighter than other commercially available near IR dyes such asLS288, IR800, and ZW800.

Example 9 Dose Escalating Studies of OTL-0038 in Mice Bearing FolateReceptor Positive Tumor Xenografts

Dosage range experiments were performed to determine the lowest dose ofOTL-0038 that can be administered to obtain best tumor-to-backgroundratio. In addition, experiments were performed to determine the highestdose of OTL-0038 that can be administered to obtain best tumor(targeted)-to-non-targeted tissue ratio.

A. Material and Methods

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude (nu/nu) mice (5 weeks old, 18-20 g) were purchasedfrom Harlan Laboratories (Indianapolis, Ind.) and maintained ongamma-irradiated folate-deficient special diet (Teklad, Wis.) for atleast 2 weeks before the start of the study. Animals were housed 5/cagein a barrier, pathogen-free cloaked rack. Autoclaved tap water and foodwere given as needed. The animals were housed in a sterile environmenton a standard 12 hours light-dark cycle for the duration of the study.Mice were identified individually by ear punch. All animal procedureswere approved by Purdue Animal Care and Use Committee. Animal care andstudies were performed according to national and internationalguidelines for the humane treatment of animals.

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (3 mice/group) wereintravenously injected with increasing concentration of OTL-0038 (0.3nmol, 1 nmol, 3 nmol, 10 nmol, 30 nmol, 60 nmol, 90 nmol) in phosphatebuffered saline (100 μL). After 2.5 h, animals were euthanized by CO₂asphyxiation. Tissue biodistribution studies were performed using aCaliper IVIS Lumina II Imaging Station with Living Image 4.0 software(PerkinElmer Inc, MA). Settings for imaging:—lamp level: medium;excitation: 745 nm; emission: ICG(indocyanine green); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Whole body images (intact tumor) were taken 2.5 hours after injecting 1nmol of OTL-0038 using a Caliper IVIS Lumina II Imaging Station withLiving Image 4.0 software (PerkinElmer Inc, MA). Settings forimaging:—lamp level: medium; excitation: 745 nm; emission: ICG; epiillumination; binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

B. Results

As shown in the Table 1 and FIG. 14, all the doses had higher tumoruptake in the folate receptor positive tumors except 0.3 nmol dose. Onthe other hand, higher kidney uptake was also observed for dose range0.3-10 nmol and less kidney uptake (relative to tumor uptake) wasobserved for dose range 30-90 nmol. Moreover, higher non-specific uptakewas observed at 60 and 90 nmol doses.

C. Conclusion

Observed lower uptake in folate receptor-positive tumors (weakfluorescence intensity) at 0.3 nmol dose may be due to incompletesaturation of folate receptors on the tumor cells. On the other hand,observed higher fluorescence intensity in the kidneys may be due to theclearance of the probe through the kidneys. Moreover, the apicalmembrane of the proximal tubule of the kidney has been known to expresshigh levels of folate receptor. Dose range between 1.0-30.0 nmol showedtumor uptake and excellent tumor-to-normal tissue ratio(signal-to-background ratio). Higher kidney uptake may be due to theclearance of the probe through the kidneys (except 30 nmol dose) andexpression of folate receptor on kidneys. Dose level 60.0 nmol andbeyond shows higher non-specific uptake. However, these still have hightumor uptake and less kidney uptake (including 30 nmol dose). Lesskidney uptake may be due to the alternative clearance of the probethrough liver and gut. Therefore, OTL-0038 may be forming aggregates atthis higher concentration. We can conclude that 1.0 nmol is the lowestdose to administer to obtain good tumor-to-background ratio whilemaintaining the non-invasive aspect for tumor imaging (FIG. 22) and 30nmol as the highest dose to administer to obtain besttumor-to-background ratio.

Example 10 Whole Body Imaging and Biodistribution of OTL-0038 in MiceBearing Folate Receptor-Negative Tumor Xenografts

Whole body imaging and tissue biodistribution was performed to determinethe in vivo specificity of OTL-0038 for folate receptors. Experimentsused mice harboring a tumor that is negative for folate receptors tocharacterize the specificity of OTL-038 compound for folate receptors.

A. Material and Methods Cell Culture and Mouse Preparation

A549 cells (a alveolar basal epithelial carcinoma cell line) wereobtained from American type culture collection (Rockville, Md.) andgrown as a monolayer using 1640 RPMI medium containing (Gibco, NY) 10%heat-inactivated fetal bovine serum (Atlanta Biological, GA) and 1%penicillin streptomycin (Gibco, NY) in a 5% carbon dioxide: 95%air-humidified atmosphere at 37° C. for at least six passages beforethey were used for the assays.

Athymic female nude (nu/nu) mice (6 weeks old, 18-20 g) were purchasedfrom Harlan Laboratories (Indianapolis, Ind.) and maintained on normaldiet (Teklad, Wis.). Animals were housed 5/cage in a barrier,pathogen-free cloaked rack. Autoclaved tap water and food were given asneeded. The animals were housed in a sterile environment on a standard12 hours light-dark cycle for the duration of the study. Mice wereidentified individually by ear punch. All animal procedures wereapproved by Purdue Animal Care and Use Committee. Animal care andstudies were performed according to national and internationalguidelines for the humane treatment of animals.

Whole Body Imaging

Seven-week-old female nu/nu mice were inoculated subcutaneously withA549 cells (1.0×10⁶/mouse in RPMI1640 medium) on the shoulder. Growth ofthe tumors was measured in perpendicular directions every 2 days using acaliper (body weights were monitored on the same schedule), and thevolumes of the tumors were calculated as 0.5×L×W² (L=longest axis andW=axis perpendicular to L in millimeters). Once tumors reached between400 and 500 mm³ in volume, animals (6 mice/group) were intravenouslyinjected with 10 nmol of OTL-0038 in phosphate buffered saline (100 μL).After 2.5 h, animals were sacrificed by CO₂ asphyxiation. Whole bodyimaging (intact tumor) experiments were then performed using a CaliperIVIS Lumina II Imaging Station with Living Image 4.0 software(PerkinElmer Inc, MA). Settings for imaging:—lamp level: medium;excitation: 745 nm; emission: ICG (indocyanine green); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Tissue Biodistribution

Following whole body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission: ICG; epi illumination; binning: 4(M), FOV=12.5; f-stop=2; acquisition time=1 s.

B. Results Whole Body Imaging

As seen in the FIG. 14, OTL-0038 did not accumulated in the folatereceptor negative tumors and there was no substantial fluorescenceactivity in the other tissues except kidneys.

Invasive Tumor and Kidney Uptake

Analysis of tumor and kidney accumulation was performed on the sameanimals that were subjected whole body imaging by euthanizing eachmouse, removing their organs and imaging using IVIS imager. As weanticipated, no fluorescence was observed in folate receptor negativetumors there was high kidney uptake. Since the apical membrane of theproximal tubule of the kidney has been known to express high levels ofFR, kidney uptake is expected. Moreover, it's possible that the probesare excreted through the kidneys due to their low molecular weights andhalf-life (most of the folate conjugates have <30 min half-life).

C. Conclusion

OTL-0038 is highly specific for folate receptor.

Example 11 Evaluation of Toxicity OTL-0038 and OTL-0039 (D-Isomer ofOTL-0038) in Healthy Nude Mice

The in-vivo toxicity of OTL-0038 and OTL-0039 was characterized inhealthy mice. Mice were administered by 1 nmol or 1000× of the clinicaldose of each compound to examine toxicity of the compounds.

A. Material and Methods

Athymic female nude (nu/nu) mice (6 weeks old, 18-20 g) were purchasedfrom Harlan Laboratories (Indianapolis, Ind.) and maintained on normaldiet (Teklad, Wis.). Animals were housed 5/cage in a barrier,pathogen-free cloaked rack. Autoclaved tap water and food were given asneeded. The animals were housed in a sterile environment on a standard12 hours light-dark cycle for the duration of the study. Mice wereidentified individually by ear punch. All animal procedures wereapproved by Purdue Animal Care and Use Committee. Animal care andstudies were performed according to national and internationalguidelines for the humane treatment of animals.

Seven-week-old healthy female nude mice (5 mice/group) were administeredwith 1 μmol of freshly prepared OTL-0038 or OTL-0039 dissolved in 100 μLof phosphate buffered saline via tail vein injection on day zero. Bodyweights and clinical observations were monitored prior to dosing anddaily thereafter from day zero to 7. Any animals with a body weight lossof 20% or more over two consecutive days would be euthanized, but thiswas not necessary. The animals were euthanized by CO₂ asphyxiation onday 7 and selected tissues (brain, heart, lung, liver, spleen, kidney,stomach, small intestine, large intestine, muscle, skin) were collectedinto vials containing 4% formalin. Formalin fixed tissues were sectionedinto 10 μm thick sections and mounted onto Superfrost Plus™ slides(Fisher Scientific, Pittsburgh Pa.). After staining the slides with H&E,immunohistochemistry (IHC) analysis of the tissues was conducted todetermine to the toxicity of OTL-0038 and OTL-0039.

B. Results

Immediately after the injection of OTL-0038 or OTL-0039, the skin of theanimals became green. However, the green color disappeared within 24hours. The animals were active after administration of the test articlesand behaved normally throughout the study. As seen in FIG. 14, the bodyweights over the course of the study remained stable. According to IHCdata (FIG. 15), there were no lesions identified in any tissues.

C. Conclusion

1 μmol (1000× clinical dose) of OTL-0038 or OTL-0039 is not toxic toanimals suggesting that OTL-0038 (1 nmol) and OTL-0039 (1 nmol) will notbe toxic to human in the clinic.

Example 12 In Vitro Pharmacological Studies of Pte-TyrosineAnalogues-S0456 (Modified OTL-0031: Analogues)

Test Articles: OTL-0040 (Pte-Tyr-¹³C-S0456), OTL-0042(Pte-Tyr-²H(Deuterated)-S0456), OTL-0043 [Pte-Tyr-(OBn)-S0456], OTL-0044[Pte-N(Me)-Tyr-S0456], OTL-0045 [Pte-NHNH-Tyr-(OAc)-S0456], OTL-0046(Pte-homo-Tyr-S0456), OTL-0047 (Pte-(3-homo-Tyr-S0456), OTL-0049(Pte-Tyramine-S0456)

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate-free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates (BD Biosciences, CA) and allowed to formmonolayers over a period of 12 h. Spent medium in each well was combinedwith 10 nM of [³H]-folic acid (tritiated folic acid) in the presence ofincreasing concentration (0.1 nM-1 μM) of the test article or folic acid(Sigma-Aldrich, MO) in fresh medium (0.5 mL). After incubating for 1 hat 37° C., cells were rinsed with PBS (3×0.5 mL, Gibco, NY) to removeany unbound radioactive materials. After adding 0.25 M sodium hydroxide(0.5 mL) and incubating for 12 h at 4° C., cells were transferred intoindividual scintillation vials containing Ecolite scintillation cocktail(3.0 mL, MP Biomedicals, OH) and counted in a liquid scintillationanalyzer (Packard). The relative binding affinities were calculatedusing a plot of % cell bound radioactivity versus the log concentrationof the test article using GraphPad Prism 4.

Results:

The dissociation constants (K_(D)) derived from the studies wascalculated to be 27.6 nM, 61.7 nM, 14.8 nM, 13.8 nM, 12.8 nM, 30.2 and 8nM for compounds OTL-0040, OTL-0042-OTL-0047, OTL-0049 and folic acidrespectively. Relative binding affinities were calculated to be 0.290,0.130, 0.177, 0.580, 0.625, 0.265 and 1 for OTL-0040, OTL-0042-OTL-0047,OTL-0049 and folic acid respectively. All the test articles competedquantitatively with [³H]-folic acid.

Conclusion:

All the compounds have an affinity for folate receptor except OTL-0044and OTL-0045 and they compare moderately well with the binding affinityof folic acid. All the compounds competed well with [³H]-folic acidindicating that folate receptor constitutes the sole binding site oncancer cells and they are highly specific for folate receptor.

Example 13 Whole-Body Imaging and Biodistribution of Pte-TyrosineAnalogues-S0456

Test Articles: OTL-0043 [Pte-Tyr-(OBn)-S0456], OTL-0044[Pte-N(Me)-Tyr-S0456], OTL-0045 [Pte-NHNH-Tyr-(OAc)-S0456], OTL-0046(Pte-homo-Tyr-S0456), OTL-0047 (Pteβ-homo-Tyr-S0456), OTL-0049(Pte-Tyramine-S0456)

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate-free 1640 RPM′ medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude mice (5 weeks old, 18-20 g) were purchased fromHarlan (IN) and maintained on gamma-irradiated folate-deficient specialdiet (Teklad, Wis.) for at least 2 weeks before the start of the study.Animals were housed 5/cage in a barrier, pathogen-free cloaked rack.Autoclaved tap water and food were given as needed. The animals werehoused in a sterile environment on a standard 12 h light-dark cycle forthe duration of the study. Mice were identified individually by earpunch. All animal procedures were approved by Purdue Animal Care and UseCommittee. Animal care and studies were performed according to nationaland international guidelines for the humane treatment of animals.

Whole-Body Imaging:

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate-free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (2-3 mice/group) wereintravenously injected with 10 nmol of test article in phosphatebuffered saline (100 μL). After 2 hours, animals were euthanized by CO₂asphyxiation. Whole-body imaging (intact tumor) experiments were thenperformed using a Caliper IVIS Lumina II Imaging Station with LivingImage 4.0 software (PerkinElmer Inc, MA). Settings for imaging:—lamplevel: medium; excitation: 745 nm; emission: ICG (830 nm); epiillumination; binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Tissue Biodistribution:

Following whole-body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, and tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission:ICG (830 nm); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Results:

Whole-Body Imaging:

As seen in the FIG. 28A, OTL-0044, OTL-0046, and OTL-0047 accumulatedpredominantly in the folate receptor-positive tumors, with nosubstantial fluorescence activity in the other tissues.

Tissue Biodistribution:

Analysis of tissue biodistribution was performed on animals under thesame conditions by euthanizing each mouse, removing their organs andimaging them using an IVIS imager. As seen in the FIG. 28B, the highestfluorescence intensity was observed in FR-positive tumors and thekidneys. The kidney uptake was anticipated since the apical membrane ofthe proximal tubule of the kidney has been known to express high levelsof folate receptor. Moreover, it's possible that the probes are excretedthrough the kidneys due to their low molecular weights and half-life(most of the folate conjugates have <30 min half-life).

Conclusion:

While in vivo biodistribution demonstrated all the compounds accumulatedin the tumors and kidneys, whole-body distribution demonstrated thatOTL-0044, OTL-0046, and OTL-0047 mainly accumulated in the tumorsindicating a requirement of alpha carboxylic acid for specificity andaffinity.

Example 14 In Vitro Pharmacology Studies of OTL-0050(Pteroyl-Tyr-S0122), OTL-0051 (Pteroyl-Tyr-IRD28), and OTL-0052(Pteroyl-Tyr-Kodak)

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates (BD Biosciences, CA) and allowed to formmonolayers over a period of 12 h. Spent medium in each well was combinedwith 10 nM of [³H]-folic acid (tritiated folic acid) in the presence ofincreasing concentration (0.1 nM-1 μM) of the test article or folic acid(Sigma-Aldrich, MO) in fresh medium (0.5 mL). After incubating for 1 hat 37° C., cells were rinsed with PBS (3×0.5 mL, Gibco, NY) to removeany unbound radioactive materials. After adding 0.25 M sodium hydroxide(0.5 mL) and incubating for 12 h at 4° C., cells were transferred intoindividual scintillation vials containing Ecolite scintillation cocktail(3.0 mL, MP Biomedicals, OH) and counted in a liquid scintillationanalyzer (Packard). The relative binding affinities were calculatedusing a plot of % cell bound radioactivity versus the log concentrationof the test article using GraphPad Prism 4.

Results:

The dissociation constants (K_(D)) derived from the studies wascalculated to be 29.3 nM, 13.8 nM, 15.3 nM, and 7.4 nM for compoundsOTL-050-OTL-0052 and folic acid respectively. Relative bindingaffinities were calculated to be 0.25, 0.54, 0.48 and 1 forOTL-0050-OTL-0052 and folic acid respectively. All the test articlescompeted quantitatively with [³H]-folic acid.

Conclusion:

OTL-0050, OTL-0051, and OTL-0052 each have an affinity for folatereceptor and the compounds compare moderately well with the bindingaffinity of folic acid. All the compounds competed well with [³H]-folicacid indicating that folate receptor constitutes the sole binding siteon cancer cells and they are highly specific for folate receptor.

Example 15 In Vitro Pharmacology Studies of Pteroyl-Non Amino Add-NR DyeConjugates

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates (BD Biosciences, CA) and allowed to formmonolayers over a period of 12 h. Spent medium in each well was combinedwith 10 nM of [³H]-folic acid (tritiated folic acid) in the presence ofincreasing concentration (0.1 nM-1 μM) of the test article or folic acid(Sigma-Aldrich, MO) in fresh medium (0.5 mL). After incubating for 1 hat 37° C., cells were rinsed with PBS (3×0.5 mL, Gibco, NY) to removeany unbound radioactive materials. After adding 0.25 M sodium hydroxide(0.5 mL) and incubating for 12 h at 4° C., cells were transferred intoindividual scintillation vials containing Ecolite scintillation cocktail(3.0 mL, MP Biomedicals, OH) and counted in a liquid scintillationanalyzer (Packard). The relative binding affinities were calculatedusing a plot of % cell bound radioactivity versus the log concentrationof the test article using GraphPad Prism 4.

Results:

The dissociation constants (K_(D)) derived from the studies wascalculated to be 95.2 nM, 121.3 nM, 90.2 nM, 250.5 nM, 225.8 nM, 41.7 nMfor compounds OTL-0056 (Pteroyl-DAP-S0456), OTL-0057(Pteroyl-BAMB-S0456), OTL-0058 (Pteroyl-AMHMB-S0456), OTL-0059(Pteroyl-DHDADS-S0456), OTL-0060 (Pteroyl-DADS-S0456), OTL-0061(Pteroyl-4APEP-S0456) and folic acid respectively. Relative bindingaffinities were calculated to be 0.078, 0.061, 0.082, 0.029, 0.033,0.171 and 1 for OTL-0056-OTL-0061 and folic acid respectively. All thetest articles competed quantitatively with [³H]-folic acid.

Conclusion:

Compounds OTL-0056-OTL-0061 each have an affinity for folate receptorand they compare moderately well with the binding affinity of folicacid. All the compounds competed well with [³H]-folic acid indicatingthat folate receptor constitutes the sole binding site on cancer cellsand they are highly specific for folate receptor.

Example 16 In Vitro Pharmacological Studies of Pteroyl-Non AminoAcid-NIR Dye Conjugates

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate-free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

KB cells that overexpress FR-α were seeded in 24-well (100,000cells/well) Falcon plates (BD Biosciences, CA) and allowed to formmonolayers over a period of 12 h. Spent medium in each well was combinedwith 10 nM of [³H]-folic acid (tritiated folic acid) in the presence ofincreasing concentration (0.1 nM-1 μM) of the test article or folic acid(Sigma-Aldrich, MO) in fresh medium (0.5 mL). After incubating for 1 hat 37° C., cells were rinsed with PBS (3×0.5 mL, Gibco, NY) to removeany unbound radioactive materials. After adding 0.25 M sodium hydroxide(0.5 mL) and incubating for 12 h at 4° C., cells were transferred intoindividual scintillation vials containing Ecolite scintillation cocktail(3.0 mL, MP Biomedicals, OH) and counted in a liquid scintillationanalyzer (Packard). The relative binding affinities were calculatedusing a plot of % cell bound radioactivity versus the log concentrationof the test article using GraphPad Prism 4.

Results:

The dissociation constants (K_(D)) derived from the studies werecalculated and found to be 95.2 nM, 121.3 nM, 90.2 nM, 250.5 nM, 225.8nM, 41.7 nM and 7.4 nM for compounds OTL-0056-OTL-0061 and folic acidrespectively. Relative binding affinities were calculated and found tobe 0.078, 0.061, 0.082, 0.029, 0.033, 0.171 and 1 for OTL-0056(Pte-DAP-S0456), OTL-0057 (Pte-BAMB-S0456), OTL-0058(Pteroyl-AMHMB-S0456), OTL-0059 (Pte-DHDADS-S0456), OTL-0060(Pte-DADS-S0456), OTL-0061 (Pte-4APEP-S0456) and folic acidrespectively. All the test articles competed quantitatively with[³H]-folic acid.

Conclusion:

All the compounds have a weak affinity for folate receptor. All thecompounds competed with [³H]-folic acid indicating that folate receptorconstitutes the sole binding site on cancer cells and they are highlyspecific for folate receptor.

Example 17 Whole-Body Imaging and Biodistribution of Pteroyl-AminoAcid-NIR Dye Conjugates

Test Articles: OTL-0038 (Pte-Tyr-S0456), OTL-0053 (Pteroyl-Lys-S0456),and OTL-0054 (Pteroyl-Cys-S0456)

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate-free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude mice (5 weeks old, 18-20 g) were purchased fromHarlan (IN) and maintained on gamma-irradiated folate-deficient specialdiet (Teklad, Wis.) for at least 2 weeks before the start of the study.Animals were housed 5/cage in a barrier, pathogen-free cloaked rack.Autoclaved tap water and food were given as needed. The animals werehoused in a sterile environment on a standard 12 h light-dark cycle forthe duration of the study. Mice were identified individually by earpunch. All animal procedures were approved by Purdue Animal Care and UseCommittee. Animal care and studies were performed according to nationaland international guidelines for the humane treatment of animals.

Whole-Body Imaging:

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate-free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (2-3 mice/group) wereintravenously injected with 10 nmol of test article in phosphatebuffered saline (100 μL). After 2 hours, animals were euthanized by CO₂asphyxiation. Whole-body (intact tumor) images were then taken using aCaliper IVIS Lumina II Imaging Station with Living Image 4.0 software(PerkinElmer Inc, MA). Settings for imaging:—lamp level: medium;excitation: 745 nm; emission: ICG (830 nm); epi illumination; binning: 4(M), FOV=12.5; f-stop=2; acquisition time=1 s. In the case ofPte-Lys-S0456, excitation: 745 nm, 710 nm, 675 nm, 640 nm, 605 nm;emission: ICG and rest of the parameters are same.

Tissue Biodistribution:

Following whole-body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, and tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission:ICG (830 nm); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Results:

Whole-Body Imaging:

As seen in the FIG. 29, OTL-0038 (Pte-Tyr-S0456) and OTL-0054(Pte-Cys-S0456) accumulated predominantly in the folatereceptor-positive tumors, with no substantial fluorescence activity inthe other tissues. Moreover, direct comparison demonstrated that tumorfluorescence intensity of OTL-0038 injected mice were brighter than themice treated with the other conjugates. On the other hand, Pte-Lys-S0456had bright tumor fluorescence when excited at 605-675 nm wave lengthsand emitted at ICG (830 nm) with only a moderate brightness when excitedat 710-745 nm and emitted at ICG, as seen in FIG. 30 (same parametersused with the other conjugates).

Tissue Biodistribution:

Analysis of tissue biodistribution was performed on animals under thesame conditions by euthanizing each mouse, removing their organs andimaging them using an IVIS imager. As seen in the FIG. 31, the highestfluorescence intensity was observed in FR-positive tumors and thekidneys. The kidney uptake was anticipated since the apical membrane ofthe proximal tubule of the kidney has been known to express high levelsof folate receptor. Moreover, it's possible that the probes are excretedthrough the kidneys due to their low molecular weights and half-life(most of the folate conjugates have <30 min half-life).

Conclusion:

The brightness and specificity of the conjugates listed from best toworst are as follows at Ex=745 nm and Em=ICG: Pte-Tyr-S0456,Pte-Cys-S0456, and Pte-Lys-S0456. Pte-Lys-S0456 all showed a longerStoke's shift, indicating that we can excite at 605 nm and emit at ICG(830 nm) to observe a bright tumor fluorescence.

Example 18 Whole-Body Imaging and Biodistribution of Pte-Tyr-KodakDerivatives [OTL-0051 (Pteroyl-Tyr-IRD28), and OTL-0052(Pteroyl-Tyr-Kodak)]

Test Articles: OTL-0051 (Pteroyl-Tyr-IRD28), and OTL-0052(Pteroyl-Tyr-Kodak)

Material and Methods:

KB cells (a human nasopharyngeal cell line) were obtained from Americantype culture collection (Rockville, Md.) and grown as a monolayer usingfolate-free 1640 RPMI medium containing (Gibco, NY) 10% heat-inactivatedfetal bovine serum (Atlanta Biological, GA) and 1% penicillinstreptomycin (Gibco, NY) in a 5% carbon dioxide: 95% air-humidifiedatmosphere at 37° C. for at least six passages before they were used forthe assays.

Athymic female nude mice (5 weeks old, 18-20 g) were purchased fromHarlan (IN) and maintained on gamma-irradiated folate-deficient specialdiet (Teklad, Wis.) for at least 2 weeks before the start of the study.Animals were housed 5/cage in a barrier, pathogen-free cloaked rack.Autoclaved tap water and food were given as needed. The animals werehoused in a sterile environment on a standard 12 h light-dark cycle forthe duration of the study. Mice were identified individually by earpunch. All animal procedures were approved by Purdue Animal Care and UseCommittee. Animal care and studies were performed according to nationaland international guidelines for the humane treatment of animals.

Whole-Body Imaging:

Seven-week-old female nu/nu mice were inoculated subcutaneously with KBcells (1.0×10⁶/mouse in folate free RPMI1640 medium) on the shoulder.Growth of the tumors was measured in perpendicular directions every 2days using a caliper (body weights were monitored on the same schedule),and the volumes of the tumors were calculated as 0.5×L×W² (L=longestaxis and W=axis perpendicular to L in millimeters). Once tumors reachedbetween 400 and 500 mm³ in volume, animals (2-3 mice/group) wereintravenously injected with 10 nmol of test article in phosphatebuffered saline (100 μL). After 2 hours, animals were euthanized by CO₂asphyxiation. Whole-body imaging (intact tumor) experiments were thenperformed using a Caliper IVIS Lumina II Imaging Station with LivingImage 4.0 software (PerkinElmer Inc, MA). Settings for imaging:—lamplevel: medium; excitation: 745 nm; emission: ICG (830 nm); epiillumination; binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.In the case of Pte-Lys-S0456, excitation: 745 nm, 710 nm, 675 nm, 640nm, 605 nm; emission: ICG (830 nm) and rest of the parameters are same.

Tissue Biodistribution:

Following whole-body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, and tumor) were analyzed for fluorescenceactivity using IVIS imager as before. Settings for imaging:—lamp level:medium; excitation: 745 nm; emission:ICG (830 nm); epi illumination;binning: 4 (M), FOV=12.5; f-stop=2; acquisition time=1 s.

Results:

Whole-Body Imaging:

As seen in the FIG. 32, OTL-0051 (Pteroyl-Tyr-IRD28), and OTL-0052(Pteroyl-Tyr-Kodak) accumulated moderately well in the folatereceptor-positive tumors, with no substantial fluorescence activity inthe other tissues. While the Kodak dye excited at 800 nM, IVIS imagesystem does not have filter to excite at 800 nM. Therefore, the lowobserved fluorescence uptake in the tumors may be due to the use of apoor excitation wavelength.

Tissue Biodistribution:

Analysis of tissue biodistribution was performed on animals under thesame conditions by euthanizing each mouse, removing their organs andimaging them using an IVIS imager. As seen in the FIG. 33, the highestfluorescence intensity was observed in FR-positive tumors. We alsoobserved uptake in the lungs. Although we anticipated having kidneyuptake (since the apical membrane of the proximal tubule of the kidneyhas been known to express high levels of folate receptor), the kidneyuptake was low.

Conclusion:

Tissue biodistribution studies demonstrated that OTL-0051(Pteroyl-Tyr-IRD28), and OTL-0052 (Pteroyl-Tyr-Kodak) conjugates uptakein the folate receptor positive tumors.

1-69. (canceled)
 70. A kit comprising a compound having the formula:

or a pharmaceutically acceptable salt thereof, or isotopes thereof,wherein: X is an single amino acid or a single amino acid derivativethereof, wherein the single amino acid or single amino acid derivativecontains an —OH, —NH2, or —SH functional group, and Y is a dye that hasa fluorescence excitation and emission spectra in the near infra redrange, wherein Y is represented by the formula:

wherein, X′ is independently selected from the group consisting of O, S,N and C, and R′ is independently selected from the group consisting ofCH₂ and CH₂CH₂. and the compound maintains or enhances the fluorescenceof the dye, Y.
 71. The kit of claim 70 wherein the amino acid isselected from the group consisting of tyrosine, cysteine, lysine, serineor a derivative thereof of tyrosine or cysteine.
 72. The kit of claim 70wherein the amino acid is tyrosine, wherein the aromatic ring oftyrosine comprises a carbon isotope and/or a hydrogen isotope.
 73. Thekit of claim 70 wherein the amino acid derivative is a derivative oftyrosine selected from the group consisting of:

or racemic mixtures thereof.
 74. The kit of claim 70 wherein the aminoacid including a sulfur-containing side chain group is cysteine.
 75. Thekit of claim 70 wherein the compound has the formula:

or is a potassium, sodium, or ammonium salt thereof.
 76. The kit ofclaim 70 wherein the compound has the formula:

or a pharmaceutically acceptable salt thereof.
 77. The kit of claim 70wherein the compound has an absorption and emission maxima between about600 nm and 800 nm.
 78. The kit of claim 70 wherein the compound is madeto fluoresce after distribution thereof in the tissue cells.
 79. The kitof claim 70 wherein the compound is highly selective for targeting to atumor cell.
 80. The kit of claim 70, wherein the kit further comprises apharmaceutically acceptable carrier, excipient or diluent.